Recombinant micro-organism for use in method with increased product yield

ABSTRACT

The invention relates to a recombinant yeast cell, in particular a transgenic yeast cell, functionally expressing one or more recombinant, in particular heterologous, nucleic acid sequences encoding ribulose-1,5-biphosphate carboxylase oxygenase (Rubisco) and phosphoribulokinase (PRK). The invention further relates to the use of carbon dioxide as an electron acceptor in a recombinant chemotrophic micro-organism, in particular a eukaryotic micro-organism.

The invention relates to a recombinant micro-organism having the abilityto produce a desired fermentation product, to the functional expressionof heterologous peptides in a micro-organism, and to a method forproducing a fermentation product wherein said microorganism is used. Ina preferred embodiment the micro-organism is a yeast. The invention isfurther related to a use of CO₂ in micro-organisms.

Microbial fermentation processes are applied for industrial productionof a broad and rapidly expanding range of chemical compounds fromrenewable carbohydrate feedstocks.

Especially in anaerobic fermentation processes, redox balancing of thecofactor couple NADH/NAD⁺ can cause important constraints on productyields. This challenge is exemplified by the formation of glycerol asmajor by-product in the industrial production of—for instance—fuelethanol by Saccharomyces cerevisiae, a direct consequence of the need toreoxidize NADH formed in biosynthetic reactions.

Ethanol production by Saccharomyces cerevisiae is currently, by volume,the single largest fermentation process in industrial biotechnology, butvarious other compounds, including other alcohols, carboxylic acids,isoprenoids, amino acids etc, are currently produced in industrialbiotechnological processes.

Various approaches have been proposed to improve the fermentativeproperties of organisms used in industrial biotechnology by geneticmodification.

WO 2008/028019 relates to a method for forming fermentation productsutilizing a microorganism having at least one heterologous genesequence, the method comprising the steps of converting at least onecarbohydrate to 3-phosphoglycerate and fixing carbon dioxide, wherein atleast one of said steps is catalyzed by at least one exogenous enzyme.Further, it relates to a microorganism for forming fermentation productsthrough fermentation of at least one sugar, the microorganism comprisingat least one heterologous gene sequence encoding at least one enzymeselected from the group consisting of phosphopentose epimerase,phosphoribulokinase, and ribulose bisphosphate carboxylase.

In an example, a yeast is mentioned wherein a heterologous PRK and aheterologous Rubisco gene are incorporated. In an embodiment the yeastis used for ethanol production. The results (FIG. 24) showconcentrations for transgenic controls and the modified strains. Littledifference is noticeable between modified yeast and its correspondingcontrol. No information is apparent regarding product yield, sugarconversion, yeast growth, evaporation rates of ethanol. Thus, it isapparent that results are not conclusive with respect to an improvementin ethanol yield.

Further, WO 2008/028019 is silent on the problem of glycerolside-product formation.

A major challenge relating to the stoichiometry of yeast-basedproduction of ethanol, but also of other compounds, is that substantialamounts of NADH-dependent side-products (in particular glycerol) aregenerally formed as a by-product, especially under anaerobic andoxygen-limited conditions or under conditions where respiration isotherwise constrained or absent. It has been estimated that, in typicalindustrial ethanol processes, up to about 4 wt. % of the sugar feedstockis converted into glycerol (Nissen et al. Yeast 16 (2000) 463-474).Under conditions that are ideal for anaerobic growth, the conversioninto glycerol may even be higher, up to about 10%.

Glycerol production under anaerobic conditions is primarily linked toredox metabolism. During anaerobic growth of S. cerevisiae, sugardissimilation occurs via alcoholic fermentation. In this process, theNADH formed in the glycolytic glyceraldehyde-3-phosphate dehydrogenasereaction is reoxidized by converting acetaldehyde, formed bydecarboxylation of pyruvate to ethanol via NAD⁺-dependent alcoholdehydrogenase. The fixed stoichiometry of this redox-neutraldissimilatory pathway causes problems when a net reduction of NAD⁺ toNADH occurs elsewhere in metabolism. Under anaerobic conditions, NADHreoxidation in S. cerevisiae is strictly dependent on reduction of sugarto glycerol. Glycerol formation is initiated by reduction of theglycolytic intermediate dihydroxyacetone phosphate (DHAP) to glycerol3-phosphate (glycerol-3P), a reaction catalyzed by NAD⁺-dependentglycerol 3-phosphate dehydrogenase. Subsequently, the glycerol3-phosphate formed in this reaction is hydrolysed byglycerol-3-phosphatase to yield glycerol and inorganic phosphate.Consequently, glycerol is a major by-product during anaerobic productionof ethanol by S. cerevisiae, which is undesired as it reduces overallconversion of sugar to ethanol. Further, the presence of glycerol ineffluents of ethanol production plants may impose costs for waste-watertreatment.

In WO 2011/010923, the NADH-related side-product (glycerol) formation ina process for the production of ethanol from a carbohydrate containingfeedstock—in particular a carbohydrate feedstock derived fromlignocellulosic biomass-glycerol side-production problem is addressed byproviding a recombinant yeast cell comprising one or more recombinantnucleic acid sequences encoding an NAD⁺-dependent acetylatingacetaldehyde dehydrogenase (EC 1.2.1.10) activity, said cell eitherlacking enzymatic activity needed for the NADH-dependent glycerolsynthesis or the cell having a reduced enzymatic activity with respectto the NADH-dependent glycerol synthesis compared to its correspondingwild-type yeast cell. A cell is described that is effective inessentially eliminating glycerol production. Also, the cell uses acetateto reoxidise NADH, whereby ethanol yield can be increased if anacetate-containing feedstock is used.

Although the described process in WO 2011/010923 is advantageous, thereis a continuing need for alternatives, in particular alternatives thatalso allow the production of a useful organic compound, such as ethanol,without needing acetate or other organic electron acceptor molecules inorder to eliminate or at least reduce NADH-dependent side-productsynthesis. It would in particular be desirable to provide amicroorganism wherein NADH-dependent side-product synthesis is reducedand which allows increased product yield, also in the absence ofacetate.

The inventors realised that it may be possible to reduce or eveneliminate NADH-dependent side-product synthesis by functionallyexpressing a recombinant enzyme in a heterotrophic, chemotrophicmicroorganism cell, in particular a yeast cell, using carbon dioxide asa substrate.

Accordingly, the present invention relates to the use of carbon dioxideas an electron acceptor in a recombinant chemoheterotrophicmicro-organism, in particular a eukaryotic micro-organism. Chemotrophic,(chemo)heterotrophic and autotrophic and other classifications of amicroorganism are herein related to the micro-organism beforerecombination, this organism is herein also referred to as the host. Forinstance, through recombination as disclosed herein a hostmicro-organism that is originally (chemo)heterotroph and not autotrophicmay become autotrophic after recombination, since applying what isdisclosed herein causes that the recombined organism may assimilatecarbon dioxide, thus resulting in (partial) (chemo) autotrophy.

Advantageously, the inventors have found a way to incorporate the carbondioxide as a co-substrate in metabolic engineering of heterotrophicindustrial microorganisms that can be used to improve product yieldsand/or to reduce side-product formation.

In particular, the inventors found it to be possible to reduce or eveneliminate NADH-dependent side-product synthesis by functionallyexpressing at least two recombinant enzyme from two specific groups in aeukaryotic microorganism, in particular a yeast cell, wherein one of theenzymes catalysis a reaction wherein carbon dioxide is used and theother uses ATP as a cofactor.

Accordingly, the invention further relates to a recombinant, in aparticular transgenic, eukaryotic microorganism, in particular a yeastcell, said microorganism functionally expressing one or morerecombinant, in particular heterologous, nucleic acid sequences encodinga ribulose-1,5-biphosphate carboxylase oxygenase (Rubisco) and aphosphoribulokinase (PRK).

A microorganism according to the invention has in particular been foundadvantageous in that in the presence of Rubisco and the PRKNADH-dependent side-product formation (glycerol) is reduced considerablyor essentially completely eliminated and production of the desiredproduct can be increased. It is thought that the carbon dioxide acts asan electron acceptor for NADH whereby less NADH is available for thereaction towards the side-product (such as glycerol).

The invention further relates to a method for preparing an organiccompound, in particular an alcohol, organic acid or amino acid,comprising converting a carbon source, in particular a carbohydrate oranother organic carbon source using a microorganism, thereby forming theorganic compound, wherein the microorganism is a microorganism accordingto the invention or wherein carbon dioxide is used as an electronacceptor in a recombinant chemotrophic or chemoheterotrophicmicro-organism.

The invention further relates to a vector for the functional expressionof a heterologous polypeptide in a yeast cell, wherein said vectorcomprises a heterologous nucleic acid sequence encoding Rubisco and PRK,wherein said Rubisco exhibits activity of carbon fixation. The term “a”or “an” as used herein is defined as “at least one” unless specifiedotherwise.

When referring to a noun (e.g. a compound, an additive, etc.) in thesingular, the plural is meant to be included. Thus, when referring to aspecific moiety, e.g. “compound”, this means “at least one” of thatmoiety, e.g. “at least one compound”, unless specified otherwise.

The term ‘or’ as used herein is to be understood as ‘and/or’.

When referring to a compound of which several isomers exist (e.g. a Dand an L enantiomer), the compound in principle includes allenantiomers, diastereomers and cis/trans isomers of that compound thatmay be used in the particular method of the invention; in particularwhen referring to such as compound, it includes the natural isomer(s).

For the purpose of clarity and a concise description features aredescribed herein as part of the same or separate embodiments, however,it will be appreciated that the scope of the invention may includeembodiments having combinations of all or some of the featuresdescribed”. In view of this passage it is evident to the skilled readerthat the variants of claim 1 as filed may be combined with otherfeatures described in the application as filed, in particular withfeatures disclosed in the dependent claims, such claims usually relatingto the most preferred embodiments of an invention.

The term ‘fermentation’, ‘fermentative’ and the like is used herein in aclassical sense, i.e. to indicate that a process is or has been carriedout under anaerobic conditions. Anaerobic conditions are herein definedas conditions without any oxygen or in which essentially no oxygen isconsumed by the yeast cell, in particular a yeast cell, and usuallycorresponds to an oxygen consumption of less than 5 mmol/l.h, inparticular to an oxygen consumption of less than 2.5 mmol/l.h, or lessthan 1 mmol/l.h. More preferably 0 mmol/L/h is consumed (i.e. oxygenconsumption is not detectable. This usually corresponds to a dissolvedoxygen concentration in the culture broth of less than 5% of airsaturation, in particular to a dissolved oxygen concentration of lessthan 1% of air saturation, or less than 0.2% of air saturation.

The term “yeast” or “yeast cell” refers to a phylogenetically diversegroup of single-celled fungi, most of which are in the division ofAscomycota and Basidiomycota. The budding yeasts (“true yeasts”) areclassified in the order Saccharomycetales, with Saccharomyces cerevisiaeas the most well known species.

The term “recombinant (cell)” or “recombinant micro-organism” as usedherein, refers to a strain (cell) containing nucleic acid which is theresult of one or more genetic modifications using recombinant DNAtechnique(s) and/or another mutagenic technique(s). In particular arecombinant cell may comprise nucleic acid not present in acorresponding wild-type cell, which nucleic acid has been introducedinto that strain (cell) using recombinant DNA techniques (a transgeniccell), or which nucleic acid not present in said wild-type is the resultof one or more mutations—for example using recombinant DNA techniques oranother mutagenesis technique such as UV-irradiation—in a nucleic acidsequence present in said wild-type (such as a gene encoding a wild-typepolypeptide) or wherein the nucleic acid sequence of a gene has beenmodified to target the polypeptide product (encoding it) towards anothercellular compartment. Further, the term “recombinant (cell)” inparticular relates to a strain (cell) from which DNA sequences have beenremoved using recombinant DNA techniques.

The term “transgenic (yeast) cell” as used herein, refers to a strain(cell) containing nucleic acid not naturally occurring in that strain(cell) and which has been introduced into that strain (cell) usingrecombinant DNA techniques, i.e. a recombinant cell).

The term “mutated” as used herein regarding proteins or polypeptidesmeans that at least one amino acid in the wild-type or naturallyoccurring protein or polypeptide sequence has been replaced with adifferent amino acid, inserted or deleted from the sequence viamutagenesis of nucleic acids encoding these amino acids. Mutagenesis isa well-known method in the art, and includes, for example, site-directedmutagenesis by means of PCR or via oligonucleotide-mediated mutagenesisas described in Sambrook et al., Molecular Cloning—A Laboratory Manual,2nd ed., Vol. 1-3 (1989). The term “mutated” as used herein regardinggenes means that at least one nucleotide in the nucleic acid sequence ofthat gene or a regulatory sequence thereof, has been replaced with adifferent nucleotide, or has been deleted from the sequence viamutagenesis, resulting in the transcription of a protein sequence with aqualitatively of quantitatively altered function or the knock-out ofthat gene.

The term “gene”, as used herein, refers to a nucleic acid sequencecontaining a template for a nucleic acid polymerase, in eukaryotes, RNApolymerase II. Genes are transcribed into mRNAs that are then translatedinto protein.

The term “nucleic acid” as used herein, includes reference to adeoxyribonucleotide or ribonucleotide polymer, i.e. a polynucleotide, ineither single or double-stranded form, and unless otherwise limited,encompasses known analogues having the essential nature of naturalnucleotides in that they hybridize to single-stranded nucleic acids in amanner similar to naturally occurring nucleotides (e.g., peptide nucleicacids). A polynucleotide can be full-length or a subsequence of a nativeor heterologous structural or regulatory gene. Unless otherwiseindicated, the term includes reference to the specified sequence as wellas the complementary sequence thereof. Thus, DNAs or RNAs with backbonesmodified for stability or for other reasons are “polynucleotides” asthat term is intended herein. Moreover, DNAs or RNAs comprising unusualbases, such as inosine, or modified bases, such as tritylated bases, toname just two examples, are polynucleotides as the term is used herein.It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including among other things,simple and complex cells.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The essential nature of such analogues of naturally occurringamino acids is that, when incorporated into a protein, that protein isspecifically reactive to antibodies elicited to the same protein butconsisting entirely of naturally occurring amino acids. The terms“polypeptide”, “peptide” and “protein” are also inclusive ofmodifications including, but not limited to, glycosylation, lipidattachment, sulphation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation.

When an enzyme is mentioned with reference to an enzyme class (EC), theenzyme class is a class wherein the enzyme is classified or may beclassified, on the basis of the Enzyme Nomenclature provided by theNomenclature Committee of the International Union of Biochemistry andMolecular Biology (NC-IUBMB), which nomenclature may be found athttp://www.chem.qmul.ac.uk/iubmb/enzyme/. Other suitable enzymes thathave not (yet) been classified in a specified class but may beclassified as such, are meant to be included.

If referred herein to a protein or a nucleic acid sequence, such as agene, by reference to a accession number, this number in particular isused to refer to a protein or nucleic acid sequence (gene) having asequence as can be found via www.ncbi.nlm.nih.gov/, (as available on 13Jul. 2009) unless specified otherwise.

Every nucleic acid sequence herein that encodes a polypeptide also, byreference to the genetic code, describes every possible silent variationof the nucleic acid. The term “conservatively modified variants” appliesto both amino acid and nucleic acid sequences. With respect toparticular nucleic acid sequences, conservatively modified variantsrefers to those nucleic acids which encode identical or conservativelymodified variants of the amino acid sequences due to the degeneracy ofthe genetic code. The term “degeneracy of the genetic code” refers tothe fact that a large number of functionally identical nucleic acidsencode any given protein. For instance, the codons GCA, GCC, GCG and GCUall encode the amino acid alanine Thus, at every position where analanine is specified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations” and represent onespecies of conservatively modified variation.

The term “functional homologue” (or in short “homologue”) of apolypeptide having a specific sequence (e.g. SEQ ID NO: X), as usedherein, refers to a polypeptide comprising said specific sequence withthe proviso that one or more amino acids are substituted, deleted,added, and/or inserted, and which polypeptide has (qualitatively) thesame enzymatic functionality for substrate conversion. Thisfunctionality may be tested by use of an assay system comprising arecombinant yeast cell comprising an expression vector for theexpression of the homologue in yeast, said expression vector comprisinga heterologous nucleic acid sequence operably linked to a promoterfunctional in the yeast and said heterologous nucleic acid sequenceencoding the homologous polypeptide of which enzymatic activity forconverting acetyl-Coenzyme A to acetaldehyde in the yeast cell is to betested, and assessing whether said conversion occurs in said cells.Candidate homologues may be identified by using in silico similarityanalyses. A detailed example of such an analysis is described in Example2 of WO2009/013159. The skilled person will be able to derive there fromhow suitable candidate homologues may be found and, optionally uponcodon (pair) optimization, will be able to test the requiredfunctionality of such candidate homologues using a suitable assay systemas described above. A suitable homologue represents a polypeptide havingan amino acid sequence similar to a specific polypeptide of more than50%, preferably of 60% or more, in particular of at least 70%, more inparticular of at least 80%, at least 90%, at least 95%, at least 97%, atleast 98% or at least 99% and having the required enzymaticfunctionality. With respect to nucleic acid sequences, the termfunctional homologue is meant to include nucleic acid sequences whichdiffer from another nucleic acid sequence due to the degeneracy of thegenetic code and encode the same polypeptide sequence.

Sequence identity is herein defined as a relationship between two ormore amino acid (polypeptide or protein) sequences or two or morenucleic acid (polynucleotide) sequences, as determined by comparing thesequences. Usually, sequence identities or similarities are comparedover the whole length of the sequences compared. In the art, “identity”also means the degree of sequence relatedness between amino acid ornucleic acid sequences, as the case may be, as determined by the matchbetween strings of such sequences.

Amino acid or nucleotide sequences are said to be homologous whenexhibiting a certain level of similarity. Two sequences being homologousindicate a common evolutionary origin. Whether two homologous sequencesare closely related or more distantly related is indicated by “percentidentity” or “percent similarity”, which is high or low respectively.Although disputed, to indicate “percent identity” or “percentsimilarity”, “level of homology” or “percent homology” are frequentlyused interchangeably. A comparison of sequences and determination ofpercent identity between two sequences can be accomplished using amathematical algorithm. The skilled person will be aware of the factthat several different computer programs are available to align twosequences and determine the homology between two sequences (Kruskal, J.B. (1983) An overview of sequence comparison In D. Sankoff and J. B.Kruskal, (ed.), Time warps, string edits and macromolecules: the theoryand practice of sequence comparison, pp. 1-44 Addison Wesley). Thepercent identity between two amino acid sequences can be determinedusing the Needleman and Wunsch algorithm for the alignment of twosequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48,443-453). The algorithm aligns amino acid sequences as well asnucleotide sequences. The Needleman-Wunsch algorithm has beenimplemented in the computer program NEEDLE. For the purpose of thisinvention the NEEDLE program from the EMBOSS package was used (version2.8.0 or higher, EMBOSS: The European Molecular Biology Open SoftwareSuite (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16,(6) pp 276-277, http://emboss.bioinformatics.nl/). For proteinsequences, EBLOSUM62 is used for the substitution matrix. For nucleotidesequences, EDNAFULL is used. Other matrices can be specified. Theoptional parameters used for alignment of amino acid sequences are agap-open penalty of 10 and a gap extension penalty of 0.5. The skilledperson will appreciate that all these different parameters will yieldslightly different results but that the overall percentage identity oftwo sequences is not significantly altered when using differentalgorithms.

Global Homology Definition

The homology or identity is the percentage of identical matches betweenthe two full sequences over the total aligned region including any gapsor extensions. The homology or identity between the two alignedsequences is calculated as follows: Number of corresponding positions inthe alignment showing an identical amino acid in both sequences dividedby the total length of the alignment including the gaps. The identitydefined as herein can be obtained from NEEDLE and is labelled in theoutput of the program as “IDENTITY”.

Longest Identity Definition

The homology or identity between the two aligned sequences is calculatedas follows: Number of corresponding positions in the alignment showingan identical amino acid in both sequences divided by the total length ofthe alignment after subtraction of the total number of gaps in thealignment. The identity defined as herein can be obtained from NEEDLE byusing the NOBRIEF option and is labeled in the output of the program as“longest-identity”.

A variant of a nucleotide or amino acid sequence disclosed herein mayalso be defined as a nucleotide or amino acid sequence having one orseveral substitutions, insertions and/or deletions as compared to thenucleotide or amino acid sequence specifically disclosed herein (e.g. inde the sequence listing).

Optionally, in determining the degree of amino acid similarity, theskilled person may also take into account so-called “conservative” aminoacid substitutions, as will be clear to the skilled person. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulphur-containing sidechains is cysteine and methionine Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine. Substitutional variants of the amino acid sequencedisclosed herein are those in which at least one residue in thedisclosed sequences has been removed and a different residue inserted inits place. Preferably, the amino acid change is conservative. Preferredconservative substitutions for each of the naturally occurring aminoacids are as follows: Ala to ser; Arg to lys; Asn to gln or his; Asp toglu; Cys to ser or ala; Gln to asn; Glu to asp; Gly to pro; His to asnor gln; Ile to leu or val; Leu to ile or val; Lys to arg; gln or glu;Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trpto tyr; Tyr to trp or phe; and, Val to ile or leu.

Nucleotide sequences of the invention may also be defined by theircapability to hybridise with parts of specific nucleotide sequencesdisclosed herein, respectively, under moderate, or preferably understringent hybridisation conditions. Stringent hybridisation conditionsare herein defined as conditions that allow a nucleic acid sequence ofat least about 25, preferably about 50 nucleotides, 75 or 100 and mostpreferably of about 200 or more nucleotides, to hybridise at atemperature of about 65° C. in a solution comprising about 1 M salt,preferably 6×SSC or any other solution having a comparable ionicstrength, and washing at 65° C. in a solution comprising about 0.1 Msalt, or less, preferably 0.2×SSC or any other solution having acomparable ionic strength. Preferably, the hybridisation is performedovernight, i.e. at least for 10 hours and preferably washing isperformed for at least one hour with at least two changes of the washingsolution. These conditions will usually allow the specific hybridisationof sequences having about 90% or more sequence identity.

Moderate conditions are herein defined as conditions that allow anucleic acid sequences of at least 50 nucleotides, preferably of about200 or more nucleotides, to hybridise at a temperature of about 45° C.in a solution comprising about 1 M salt, preferably 6×SSC or any othersolution having a comparable ionic strength, and washing at roomtemperature in a solution comprising about 1 M salt, preferably 6×SSC orany other solution having a comparable ionic strength. Preferably, thehybridisation is performed overnight, i.e. at least for 10 hours, andpreferably washing is performed for at least one hour with at least twochanges of the washing solution. These conditions will usually allow thespecific hybridisation of sequences having up to 50% sequence identity.The person skilled in the art will be able to modify these hybridisationconditions in order to specifically identify sequences varying inidentity between 50% and 90%.

“Expression” refers to the transcription of a gene into structural RNA(rRNA, tRNA) or messenger RNA (mRNA) with subsequent translation into aprotein.

As used herein, “heterologous” in reference to a nucleic acid or proteinis a nucleic acid or protein that originates from a foreign species, or,if from the same species, is substantially modified from its native formin composition and/or genomic locus by deliberate human intervention.For example, a promoter operably linked to a heterologous structuralgene is from a species different from that from which the structuralgene was derived, or, if from the same species, one or both aresubstantially modified from their original form. A heterologous proteinmay originate from a foreign species or, if from the same species, issubstantially modified from its original form by deliberate humanintervention.

The term “heterologous expression” refers to the expression ofheterologous nucleic acids in a host cell. The expression ofheterologous proteins in eukaryotic host cell systems such as yeast arewell known to those of skill in the art. A polynucleotide comprising anucleic acid sequence of a gene encoding an enzyme with a specificactivity can be expressed in such a eukaryotic system. In someembodiments, transformed/transfected yeast cells may be employed asexpression systems for the expression of the enzymes. Expression ofheterologous proteins in yeast is well known. Sherman, F., et al.,Methods in Yeast Genetics, Cold Spring Harbor Laboratory (1982) is awell recognized work describing the various methods available to expressproteins in yeast. Two widely utilized yeasts are Saccharomycescerevisiae and Pichia pastoris. Vectors, strains, and protocols forexpression in Saccharomyces and Pichia are known in the art andavailable from commercial suppliers (e.g., Invitrogen). Suitable vectorsusually have expression control sequences, such as promoters, including3-phosphoglycerate kinase or alcohol oxidase, and an origin ofreplication, termination sequences and the like as desired.

As used herein “promoter” is a DNA sequence that directs thetranscription of a (structural) gene. Typically, a promoter is locatedin the 5′-region of a gene, proximal to the transcriptional start siteof a (structural) gene. Promoter sequences may be constitutive,inducible or repressible. If a promoter is an inducible promoter, thenthe rate of transcription increases in response to an inducing agent.

The term “vector” as used herein, includes reference to an autosomalexpression vector and to an integration vector used for integration intothe chromosome.

The term “expression vector” refers to a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestunder the control of (i.e. operably linked to) additional nucleic acidsegments that provide for its transcription. Such additional segmentsmay include promoter and terminator sequences, and may optionallyinclude one or more origins of replication, one or more selectablemarkers, an enhancer, a polyadenylation signal, and the like. Expressionvectors are generally derived from plasmid or viral DNA, or may containelements of both. In particular an expression vector comprises a nucleicacid sequence that comprises in the 5′ to 3′ direction and operablylinked: (a) a yeast-recognized transcription and translation initiationregion, (b) a coding sequence for a polypeptide of interest, and (c) ayeast-recognized transcription and translation termination region.“Plasmid” refers to autonomously replicating extrachromosomal DNA whichis not integrated into a microorganism's genome and is usually circularin nature.

An “integration vector” refers to a DNA molecule, linear or circular,that can be incorporated in a microorganism's genome and provides forstable inheritance of a gene encoding a polypeptide of interest. Theintegration vector generally comprises one or more segments comprising agene sequence encoding a polypeptide of interest under the control of(i.e. operably linked to) additional nucleic acid segments that providefor its transcription. Such additional segments may include promoter andterminator sequences, and one or more segments that drive theincorporation of the gene of interest into the genome of the targetcell, usually by the process of homologous recombination. Typically, theintegration vector will be one which can be transferred into the targetcell, but which has a replicon which is nonfunctional in that organism.Integration of the segment comprising the gene of interest may beselected if an appropriate marker is included within that segment.

By “host cell” is meant a cell which contains a vector and supports thereplication and/or expression of the vector. Host cells may beprokaryotic cells such as E. coli, or eukaryotic cells such as yeast,insect, amphibian, or mammalian cells. Preferably, host cells areeukaryotic cells of the order of Actinomycetales.

“Transformation” and “transforming”, as used herein, refers to theinsertion of an exogenous polynucleotide into a host cell, irrespectiveof the method used for the insertion, for example, direct uptake,transduction, f-mating or electroporation. The exogenous polynucleotidemay be maintained as a non-integrated vector, for example, a plasmid, oralternatively, may be integrated into the host cell genome.

The microorganism, preferably is selected from the group ofSaccharomyceraceae, such as Saccharomyces cerevisiae, Saccharomycespastorianus, Saccharomyces beticus, Saccharomyces fermentati,Saccharomyces paradoxus, Saccharomyces uvarum and Saccharomyces bayanus;Schizosaccharomyces such as Schizosaccharomyces pombe,Schizosaccharomyces japonicus, Schizosaccharomyces octosporus andSchizosaccharomyces cryophilus; Torulaspora such as Torulasporadelbrueckii; Kluyveromyces such as Kluyveromyces marxianus; Pichia suchas Pichia stipitis, Pichia pastoris or pichia angusta, Zygosaccharomycessuch as Zygosaccharomyces bailii; Brettanomyces such as Brettanomycesintermedius, Brettanomyces bruxellensis, Brettanomyces anomalus,Brettanomyces custersianus, Brettanomyces naardenensis, Brettanomycesnanus, Dekkera bruxellensis and Dekkera anomala; Metschnikowia,Issatchenkia, such as Issatchenkia orientalis, Kloeckera such asKloeckera apiculate; Aureobasidium such as Aureobasidium pullulans.

In a highly preferred embodiment, the microorganism is a yeast cell isselected from the group of Saccharomyceraceae. In particular, goodresults have been achieved with a Saccharomyces cerevisiae cell. It hasbeen found possible to use such a cell according to the invention in amethod for preparing an alcohol (ethanol) wherein the NADH-dependentside-product formation (glycerol) was reduced by about 90%, and whereinthe yield of the desired product (ethanol) was increase by about 10%,compared to a similar cell without Rubisco and PRK.

The Rubisco may in principle be selected from eukaryotic and prokaryoticRubisco's.

The Rubisco is preferably from a non-phototrophic organism. Inparticular, the Rubisco may be from a chemolithoautotrophicmicroorganism.

Good results have been achieved with a bacterial Rubisco. Preferably,the bacterial Rubisco originates from a Thiobacillus, in particular,Thiobacillus denitrificans, which is chemolithoautotrophic.

The Rubisco may be a single-subunit Rubisco or a Rubisco having morethan one subunit. In particular, good results have been achieved with asingle-subunit Rubisco.

In particular, good results have been achieved with a form-II Rubisco,more in particular CbbM.

SEQUENCE ID NO: 2 shows the sequence of a particularly preferred Rubiscoin accordance with the invention. It is encoded by the cbbM gene fromThiobacillus denitrificans. A preferred alternative to this Rubisco, isa functional homologue of this Rubisco, in particular such functionalhomologue comprising a sequence having at least 80%, 85%, 90% or 95%sequence identity with SEQUENCE ID NO: 2. Suitable natural Rubiscopolypeptides are given in Table 1.

TABLE 1 Rubisco polypeptides Source Accession no. MAX ID (%)Thiobacillus denitrificans AAA99178.2 100 Sideroxydans lithotrophicusES-1 YP_003522651.1 94 Thiothrix nivea DSM 5205 ZP_10101642.1 91Halothiobacillus neapolitanus c2 YP_003262978.1 90 Acidithiobacillusferrooxidans ATCC YP_002220242.1 88 53993 Rhodoferax ferrireducens T118YP_522655.1 86 Thiorhodococcus drewsii AZ1 ZP_08824342.1 85 unculturedprokaryote AGE14067.1 82

In accordance with the invention, the Rubisco is functionally expressedin the microorganism, at least during use in an industrial process forpreparing a compound of interest.

To increase the likelihood that herein enzyme activity is expressed atsufficient levels and in active form in the transformed (recombinant)host cells of the invention, the nucleotide sequence encoding theseenzymes, as well as the Rubisco enzyme and other enzymes of theinvention (see below), are preferably adapted to optimise their codonusage to that of the host cell in question. The adaptiveness of anucleotide sequence encoding an enzyme to the codon usage of a host cellmay be expressed as codon adaptation index (CAI). The codon adaptationindex is herein defined as a measurement of the relative adaptiveness ofthe codon usage of a gene towards the codon usage of highly expressedgenes in a particular host cell or organism. The relative adaptiveness(w) of each codon is the ratio of the usage of each codon, to that ofthe most abundant codon for the same amino acid. The CAI index isdefined as the geometric mean of these relative adaptiveness values.Non-synonymous codons and termination codons (dependent on genetic code)are excluded. CAI values range from 0 to 1, with higher valuesindicating a higher proportion of the most abundant codons (see Sharpand Li, 1987, Nucleic Acids Research 15: 1281-1295; also see: Jansen etal., 2003, Nucleic Acids Res. 31(8):2242-51). An adapted nucleotidesequence preferably has a CAI of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8 or 0.9. Most preferred are the sequences which have been codonoptimised for expression in the fungal host cell in question such ase.g. S. cerevisiae cells.

Preferably, the functionally expressed Rubisco has an activity, definedby the rate of ribulose-1,5-bisphosphate-dependent ¹⁴C-bicarbonateincorporation by cell extracts of at least 1 nmol min⁻¹.(mg protein)⁻¹,in particular an activity of at least 2 nmol min⁻¹.(mg protein)⁻¹, morein particular an activity of at least 4 nmol.min⁻¹.(mg protein)⁻¹. Theupper limit for the activity is not critical. In practice, the activitymay be about 200 nmol min⁻¹.(mg protein)⁻¹ or less, in particular 25nmol min⁻¹.(mg protein)⁻¹, more in particular 15 nmol min⁻¹.(mgprotein)⁻¹ or less, e.g. about 10 nmol min⁻¹.(mg protein)⁻¹ or less.When referred herein to the activity of Rubisco, in particular theactivity at 30° C. is meant. The conditions for an assay for determiningthis Rubisco activity are as found in the Examples, below (Example 4).

A functionally expressed phosphoribulokinase (PRK, (EC 2.7.1.19))according to the invention is capable of catalysing the chemicalreaction:

ATP+D-ribulose 5-phosphate⇄ADP+D-ribulose 1,5-bisphosphate  (1)

Thus, the two substrates of this enzyme are ATP and D-ribulose5-phosphate, whereas its two products are ADP and D-ribulose1,5-bisphosphate.

PRK belongs to the family of transferases, specifically thosetransferring phosphorus-containing groups (phosphotransferases) with analcohol group as acceptor. The systematic name of this enzyme class isATP:D-ribulose-5-phosphate 1-phosphotransferase. Other names in commonuse include phosphopentokinase, ribulose-5-phosphate kinase,phosphopentokinase, phosphoribulokinase (phosphorylating),5-phosphoribulose kinase, ribulose phosphate kinase, PKK, PRuK, and PRK.This enzyme participates in carbon fixation.

The PRK can be from a prokaryote or a eukaryote. Good results have beenachieved with a PRK originating from a eukaryote. Preferably theeukaryotic PRK originates from a plant selected from Caryophyllales, inparticular from Amaranthaceae, more in particular from Spinacia.

As a preferred alternative to PRK from Spinacia a functional homologueof PRK from Spinacia may be present, in particular a functionalhomologue comprising a sequence having at least 70%, 75%, 80%. 85%, 90%or 95% sequence identity with SEQUENCE ID NO 4.

Suitable natural PRK polypeptides are given in Table 2.

TABLE 2 Natural PRK polypeptides suitable for expression SourceAccession no. MAX ID (%) Spinacia oleracea P09559.1 100 Medicagotruncatula XP_003612664.1 88 Arabidopsis thaliana NP_174486.1 87 Vitisvinifera XP_002263724.1 86 Closterium peracerosum BAL03266.1 82 Zea maysNP_001148258.1 78In an advantageous embodiment, the recombinant microorganism furthercomprises a nucleic acid sequence encoding one or more heterologousprokaryotic or eukaryotic molecular chaperones, which—when expressed—arecapable of functionally interacting with an enzyme in the microorganism,in particular with at least one of Rubisco and PRK.

Chaperonins are proteins that provide favourable conditions for thecorrect folding of other proteins, thus preventing aggregation. Newlymade proteins usually must fold from a linear chain of amino acids intoa three-dimensional form. Chaperonins belong to a large class ofmolecules that assist protein folding, called molecular chaperones. Theenergy to fold proteins is supplied by adenosine triphosphate (ATP). Areview article about chaperones that is useful herein is written byYébenes (2001); “Chaperonins: two rings for folding”; Hugo Yébenes etal. Trends in Biochemical Sciences, August 2011, Vol. 36, No. 8.

In a preferred embodiment, the chaperone or chaperones are from abacterium, more preferably from Escherichia, in particular E. coli GroELand GroEs from E. coli may in particular encoded in a microorganismaccording to the invention. Other preferred chaperones are chaperonesfrom Saccharomyces, in particular Saccharomyces cerevisiae Hsp10 andHsp60. If the chaperones are naturally expressed in an organelle such asa mitochondrion (examples are Hsp60 and Hsp10 of Saccharomycescerevisiae) relocation to the cytosol can be achieved e.g. by modifyingthe native signal sequence of the chaperonins.

In eukaryotes the proteins Hsp60 and Hsp10 are structurally andfunctionally nearly identical to GroEL and GroES, respectively. Thus, itis contemplated that Hsp60 and Hsp10 from any eukaryotic cell may serveas a chaperone for the Rubisco. See Zeilstra-Ryalls J, Fayet O,Georgopoulos C (1991). “The universally conserved GroE (Hsp60)chaperonins”. Annu Rev Microbiol. 45: 301-25.doi:10.1146/annurev.mi.45.100191.001505. PMID 1683763 and Horwich A L,Fenton W A, Chapman E, Farr G W (2007). “Two Families of Chaperonin:Physiology and Mechanism”. Annu Rev Cell Dev Biol. 23: 115-45.

doi:10.1146/annurev.cellbio.23.090506.123555. PMID 17489689.

Particularly good results have been achieved with a recombinant yeastcell comprising both the heterologous chaperones GroEL and GroES.

As a preferred alternative to GroEL a functional homologue of GroEL maybe present, in particular a functional homologue comprising a sequencehaving at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity withSEQUENCE ID NO: 10.

Suitable natural chaperones polypeptide homologous to SEQUENCE ID NO: 10are given in Table 3.

TABLE 3 Natural chaperones homologous to SEQUENCE ID NO: 10 polypeptidessuitable for expression >gi | 115388105 | ref | XP_001211558.1 | :2-10110 kDa heat shock protein, mitochondrial [Aspergillus terreusNIH2624] >gi | 116196854 | ref | XP_001224239.1 | :1-102 conservedhypothetical protein [Chaetomium globosum CBS 148.51] >gi | 119175741 |ref | XP_001240050.1 | :3-102 hypothetical protein CIMG_09671[Coccidioides immitis RS] >gi | 119471607 | ref | XP_001258195.1 |:12-111 chaperonin, putative [Neosartorya fischeri NRRL181] >gi |121699818 | ref | XP_001268174.1 | :8-106 chaperonin, putative[Aspergillus clavatus NRRL 1] >gi | 126274604 | ref | XP_001387607.1 |:2-102 predicted protein [Scheffersomyces stipitis CBS 6054] >gi |146417701 | ref | XP_001484818.1 | :5-106 conserved hypothetical protein[Meyerozyma guilliermondii ATCC 6260] >gi | 154303611 | ref |XP_001552212.1 | :1-102 10 kDa heat shock protein, mitochondrial[Botryotinia fuckeliana B05.10] >gi | 156049571 | ref | XP_001590752.1 |:1-102 hypothetical protein SS1G_08492 [Sclerotinia sclerotiorum1980] >gi | 156840987 | ref | XP_001643870.1 | :1-103 hypotheticalprotein Kpol_495p10 [Vanderwaltozyma polyspora DSM 70924] >gi |169608295 | ref | XP_001797567.1 | :1-101 hypothetical proteinSNOG_07218 [Phaeosphaeria nodorum SN15] >gi | 171688384 | ref |XP_001909132.1 | :1-102 hypothetical protein [Podospora anserina Smat+] >gi | 189189366 | ref | XP_001931022.1 | :71-168 10 kDa chaperonin[Pyrenophora tritici-repentis Pt-1C-BFP] >gi | 19075598 | ref |NP_588098.1 | :1-102 mitochondrial heat shock protein Hsp10 (predicted)[Schizosaccharomyces pombe 972h-] >gi | 212530240 | ref | XP_002145277.1| :3-100 chaperonin, putative [Talaromyces marneffei ATCC 18224] >gi |212530242 | ref | XP_002145278.1 | :3-95 chaperonin, putative[Talaromyces marneffei ATCC 18224] >gi | 213404320 | ref |XP_002172932.1 | :1-102 mitochondrial heat shock protein Hsp10[Schizosaccharomyces japonicus yFS275] >gi | 225557301 | gb | EEH05587.1| :381-478 pre-mRNA polyadenylation factor fip1 [Ajellomyces capsulatusG186AR] >gi | 225684092 | gb | EEH22376.1 | :3-100 heat shock protein[Paracoccidioides brasiliensis Pb03 >gi | 238490530 | ref |XP_002376502.1 | :2-104 chaperonin, putative [Aspergillus flavusNRRL3357 >gi | 238878220 | gb | EEQ41858.1 | :1-106 10 kDa heat shockprotein, mitochondrial [Candida albicans WO-1] >gi | 240280207 | gb |EER43711.1 | :426-523 pre-mRNA polyadenylation factor fip1 [Ajellomycescapsulatus H143] >gi | 241950445 | ref | XP_002417945.1 | :1-103 10 kdachaperonin, putative; 10 kda heat shock protein mitochondrial (hsp10),putative [Candida dubliniensis CD36] >gi | 242819222 | ref |XP_002487273.1 | :90-182 chaperonin, putative [Talaromyces stipitatusATC >gi | 254566327 | ref | XP_002490274.1 | :1-102 Putative protein ofunknown function [Komagataella pastoris GS115] >gi | 254577241 | ref |XP_002494607.1 | :1-103 ZYRO0A05434p [Zygosaccharomyces rouxii] >gi |255717999 | ref | XP_002555280.1 | :1-103 KLTH0G05588p [Lachanceathermotolerans] >gi | 255956581 | ref | XP_002569043.1 | :2-101Pc21g20560 [Penicillium chrysogenum Wisconsin 54-1255] >gi | 258572664 |ref | XP_002545094.1 | :16-108 chaperonin GroS [Uncinocarpus reesii1704] >gi | 261190594 | ref | XP_002621706.1 | :3-100 chaperonin[Ajellomyces dermatitidis SLH14081] >gi | 295664909 | ref |XP_002793006.1 | :3-100 10 kDa heat shock protein, mitochondrial[Paracoccidioides sp. ‘lutzii’Pb01] >gi | 296412657 | ref |XP_002836039.1 | :76-177 hypothetical protein [Tuber melanosporumMel28] >gi | 302307854 | ref | NP_984626.2 | :2-102 AEL235Wp [Ashbyagossypii ATCC 10895] >gi | 302894117 | ref | XP_003045939.1 | :1-102predicted protein [Nectria haematococca mpVI 77-13-4] >gi | 303318351 |ref | XP_003069175.1 | :3-100 10 kDa heat shock protein, mitochondrial,putative [Coccidioides posadasii C735 delta SOWgp] >gi | 310795300 | gb| EFQ30761.1 | :1-102 chaperonin 10 kDa subunit [Glomerella graminicolaM1.001] >gi | 315053085 | ref | XP_003175916.1 | :12-109 chaperonin GroS[Arthroderma gypseum CBS 118893] >gi | 317032114 | ref | XP_001394060.2| :334-433 heat shock protein [Aspergillus niger CBS 513.88] >gi |317032116 | ref | XP_001394059.2 | :2-101 heat shock protein[Aspergillus niger CBS 513.88] >gi | 320583288 | gb | EFW97503.1 |:6-106 chaperonin, putative heat shock protein, putative [Ogataeaparapolymorpha DL-1] >gi | 320591507 | gb | EFX03946.1 | :1-102 heatshock protein [Grosmannia clavigera kw1407] >gi | 322700925 | gb |EFY92677.1 | :1-102 chaperonin [Metarhizium acridum CQMa 102] >gi |325096696 | gb | EGC50006.1 | :409-506 pre-mRNA polyadenylation factorfip1 [Ajellomyces capsulatus H88] >gi | 326471604 | gb | EGD95613.1 |:14-111 chaperonin 10 Kd subunit [Trichophyton tonsurans CBS112818] >gi| 327293056 | ref | XP_003231225.1 | :3-100 chaperonin [Trichophytonrubrum CBS 118892] >gi | 330942654 | ref | XP_003306155.1 | :37-136hypothetical protein PTT_19211 [Pyrenophora teres f. teres 0-1] >gi |336268042 | ref | XP_003348786.1 | :47-147 hypothetical proteinSMAC_01809 [Sordaria macrospora khell] >gi | 340519582 | gb | EGR49820.1| :1-109 predicted protein [Trichoderma reesei QM6a] >gi | 340960105 |gb | EGS21286.1 | :3-103 putative mitochondrial 10 kDa heat shockprotein [Chaetomium thermophilum var. thermophilum DSM 1495] >gi |342883802 | gb | EGU84224.1 | :1-102 hypothetical protein FOXB_05181[Fusarium oxysporum Fo5176] >gi | 344302342 | gb | EGW32647.1 | :2-102hypothetical protein SPAPADRAFT_61712 [Spathaspora passalidarum NRRLY-27907] >gi | 345570750 | gb | EGX53571.1 | :1-102 hypothetical proteinAOL_s00006g437 [Arthrobotrys oligospora ATCC 24927] >gi | 346321154 | gb| EGX90754.1 | :1-102 chaperonin [Cordyceps militaris CM01] >gi |346970393 | gb | EGY13845.1 | :1-102 heat shock protein [Verticilliumdahliae VdLs.17] >gi | 354548296 | emb | CCE45032.1 | :1-106hypothetical protein CPAR2_700360 [Candida parapsilosis] >gi | 358385052| gb | EHK22649.1 | :1-102 hypothetical protein TRIVIDRAFT_230640[Trichoderma virens Gv 29-8] >gi | 358393422 | gb | EHK42823.1 | :1-101hypothetical protein TRIATDRAFT_258186 [Trichoderma atroviride IMI206040] >gi | 361126733 | gb | EHK98722.1 | :1-97 putative 10 kDa heatshock protein, mitochondrial [Glare lozoyensis 74030] >gi | 363753862 |ref | XP_003647147.1 | :2-102 hypothetical protein Ecym_5593[Eremothecium cymbalariae DBVPG#7215] >gi | 365758401 | gb | EHN00244.1| :1-106 Hsp10p [Saccharomyces cerevisiae × Saccharomyces kudriavzeviiVIN7] >gi | 365987664 | ref | XP_003670663.1 | :1-103 hypotheticalprotein NDAI_0F01010 [Naumovozyma dairenensis CBS 421] >gi | 366995125 |ref | XP_003677326.1 | :1-103 hypothetical protein NCAS_0G00860[Naumovozyma castellii CBS 4309] >gi | 366999797 | ref | XP_003684634.1| :1-103 hypothetical protein TPHA_0C00430 [Tetrapisispora phaffii CBS4417] >gi | 367009030 | ref | XP_003679016.1 | :1-103 hypotheticalprotein TDEL_0A04730 [Torulaspora delbruekii] >gi | 367023138 | ref |XP_003660854.1 | :1-104 hypothetical protein MYCTH_59302 [Myceliophthorathermophila ATCC 42464] >gi | 367046344 | ref | XP_003653552.1 | :1-102hypothetical protein THITE_2116070 [Thielavia terrestris NRRL8126] >gi |378726440 | gb | EHY52899.1 | :9-109 chaperonin GroES [Exophialadermatitidis NIH/UT8656] >gi | 380493977 | emb | CCF33483.1 | :1-102chaperonin 10 kDa subunit [Colletotrichum higginsianu >gi | 385305728 |gb | EIF49680.1 | :1-102 10 kda heat shock mitochondrial [Dekkerabruxellensis AWRI1499] >gi | 389628546 | ref | XP_003711926.1 | :1-102hsp10-like protein [Magnaporthe oryzae 70-15] >gi | 396462608 | ref |XP_003835915.1 | :1-101 similar to 10 kDa heat shock protein[Leptosphaeria maculans JN3] >gi | 398392541 | ref | XP_003849730.1 |:1-102 hypothetical protein MYCGRDRAFT_105721 [Zymoseptoria triticiIPO323] >gi | 400597723 | gb | EJP65453.1 | :24-124 chaperonin 10 kDasubunit [Beauveria bassiana ARSEF 2860] >gi | 401623646 | gb |EJS41738.1 | :1-106 hsp10p [Saccharomyces arboricola H-6] >gi |401842164 | gb | EJT44422.1 | :1-92 HSP10-like protein [Saccharomyceskudriavzevii IFO 1802] >gi | 402084027 | gb | EJT79045.1 | :1-102hsp10-like protein [Gaeumannomyces graminis var. triti >gi | 403215209 |emb | CCK69709.1 | :1-104 hypothetical protein KNAG_0C06130[Kazachstania naganishii CBS 8797] >gi | 406604629 | emb | CCH43969.1 |:4-100 hypothetical protein BN7_3524 [Wickerhamomyces ciferrii] >gi |406867021 | gb | EKD20060.1 | :56-156 hypothetical protein MBM_02012[Marssonina brunnea f. sp. ‘multigermtubi’ MB_m1] >gi | 407926227 | gb |EKG19196.1 | :74-174 GroES-like protein [Macrophomina phaseolinaMS6] >gi | 408398157 | gb | EKJ77291.1 | :11-111 hypothetical proteinFPSE_02566 [Fusarium pseudograminearum CS3096] >gi | 410082063 | ref |XP_003958610.1 | :1-103 hypothetical protein KAFR_0H00660 [Kazachstaniaafricana CBS2517] >gi | 425777664 | gb | EKV15823.1 | :58-157Chaperonin, putative [Penicillium digitatum Pd1] >gi | 440639680 | gb |ELR09599.1 | :1-102 chaperonin GroES [Geomyces destructans 20631-21] >gi| 444323906 | ref | XP_004182593.1 | :1-105 hypothetical proteinTBLA_0J00760 [Tetrapisisporablattae CBS 6284] >gi | 448083208 | ref |XP_004195335.1 | :2-101 Piso0_005888 [Millerozyma farinosa CBS 7064] >gi| 448087837 | ref | XP_004196425.1 | :2-102 Piso0_005888 [Millerozymafarinosa CBS 7064] >gi | 448534948 | ref | XP_003870866.1 | :1-106 Hsp10protein [Candida orthopsilosis Co 90-125] >gi | 449295977 | gb |EMC91998.1 | :1-102 hypothetical protein BAUCODRAFT_39148 [Baudoiniacompn >gi | 46123659 | ref | XP_386383.1 | :3-103 hypothetical proteinFG06207.1 [Gibberella zeae PH-1] >gi | 50289455 | ref | XP_447159.1 |:1-103 hypothetical protein [Candida glabrata CBS 138] >gi | 50308731 |ref | XP_454370.1 | :1-103 hypothetical protein [Kluyveromyces lactisNRRL Y-1140] >gi | 50411066 | ref | XP_457014.1 | :1-106 DEHA2B01122p[Debaryomyces hansenii CBS767] >gi | 50545998 | ref | XP_500536.1 |:1-102 YALI0B05610p [Yarrowia lipolytica] >gi | 51013895 | gb |AAT93241.1 | :1-106 YOR020C [Saccharomyces cerevisiae] >gi | 6324594 |ref | NP_014663.1 | :1-106 Hsp10p [Saccharomyces cerevisiae S288c] >gi |67523953 | ref | XP_660036.1 | :2-101 hypothetical protein AN2432.2[Aspergillus nidulans FGSC A4] >gi | 70992219 | ref | XP_750958.1 |:12-106 chaperonin [Aspergillus fumigatus Af293] >gi | 85079266 | ref |XP_956315.1 | :1-104 hypothetical protein NCU04334 [Neurospora crassaOR74A]

As a preferred alternative to GroES a functional homologue of GroES maybe present, in particular a functional homologue comprising a sequencehaving at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity withSEQUENCE ID NO: 12.

Suitable natural chaperones polypeptides homologous to SEQUENCE ID NO:12 are given in Table 4.

TABLE 4 Natural chaperones homologous to SEQUENCE ID NO: 12 polypeptidessuitable for expression >gi | 115443330 | ref | XP_001218472.1 | heatshock protein 60, mitochondrial precursor [Aspergillus terreusNIH2624] >gi | 114188341 | gb | EAU30041.1 | heat shock protein 60,mitochondrial precursor [Aspergillus terreus NIH2624] >gi | 119480793 |ref | XP_001260425.1 | antigenic mitochondrial protein HSP60, putative[Neosartorya fischeri NRRL 181] >gi | 119408579 | gb | EAW18528.1 |antigenic mitochondrial protein HSP60, putative [Neosartorya fischeriNRRL 181] >gi | 126138730 | ref | XP_001385888.1 | hypothetical proteinPICST_90190 [Scheffersomyces stipitis CBS 6054] >gi | 126093166 | gb |ABN67859.1 | mitochondrial groEL-type heat shock protein[Scheffersomyces stipitis CBS 6054] >gi | 145246630 | ref |XP_001395564.1 | heat shock protein 60 [Aspergillus niger CBS513.88] >gi | 134080285 | emb | CAK46207.1 | unnamed protein product[Aspergillus niger] >gi | 350636909 | gb | EHA25267.1 | hypotheticalprotein ASPNIDRAFT_54001 [Aspergillus niger ATCC 1015] >gi | 146413148 |ref | XP_001482545.1 | heat shock protein 60, mitochondrial precursor[Meyerozyma guilliermondii ATCC 6260] >gi | 154277022 | ref |XP_001539356.1 | heat shock protein 60, mitochondrial precursor[Ajellomyces capsulatus NAm1] >gi | 150414429 | gb | EDN09794.1 | heatshock protein 60, mitochondrial precursor [Ajellomyces capsulatusNAm1] >gi | 154303540 | ref | XP_001552177.1 | heat shock protein 60[Botryotinia fuckeliana B05.10] >gi | 347840915 | emb | CCD55487.1 |similar to heat shock protein 60 [Botryotinia fuckeliana] >gi |156063938 | ref | XP_001597891.1 | heat shock protein 60, mitochondrialprecursor [Sclerotinia sclerotiorum 1980] >gi | 154697421 | gb |EDN97159.1 | heat shock protein 60, mitochondrial precursor [Sclerotiniasclerotiorum 1980 UF-70] >gi | 156844469 | ref | XP_001645297.1 |hypothetical protein Kpol_1037p35 [Vanderwaltozyma polyspora DSM70294] >gi | 156115957 | gb | EDO17439.1 | hypothetical proteinKpol_1037p35 [Vanderwaltozyma polyspora DSM 70294] >gi | 16416029 | emb| CAB91379.2 | probable heat-shock protein hsp60 [Neurospora crassa] >gi| 350289516 | gb | EGZ70741.1 | putative heat-shock protein hsp60[Neurospora tetrasperma FGSC 2509] >gi | 169626377 | ref |XP_001806589.1 | hypothetical protein SNOG_16475 [Phaeosphaeria nodorumSN15] >gi | 111055053 | gb | EAT76173.1 | hypothetical proteinSNOG_16475 [Phaeosphaeria nodorum SN15] >gi | 169783766 | ref |XP_001826345.1 | heat shock protein 60 [Aspergillus oryzae RIB40] >gi |238493601 | ref | XP_002378037.1 | antigenic mitochondrial proteinHSP60, putative [Aspergillus flavus NRRL3357] >gi | 83775089 | dbj |BAE65212.1 | unnamed protein product [Aspergillus oryzae RIB40] >gi |220696531 | gb | EED52873.1 | antigenic mitochondrial protein HSP60,putative [Aspergillus flavus NRRL3357] >gi | 391869413 | gb | EIT78611.1| chaperonin, Cpn60/Hsp60p [Aspergillus oryzae 3.042] >gi | 189190432 |ref | XP_001931555.1 | heat shock protein 60, mitochondrial precursor[Pyrenophora tritici-repentis Pt-1C-BFP] >gi | 187973161 | gb |EDU40660.1 | heat shock protein 60, mitochondrial precursor [Pyrenophoratritici-repentis Pt-1C-BFP] >gi | 190348913 | gb | EDK41467.2 | heatshock protein 60, mitochondrial precursor [Meyerozyma guilliermondiiATCC 6260] >gi | 225554633 | gb | EEH02929.1 | hsp60-like protein[Ajellomyces capsulatus G186AR] >gi | 238880068 | gb | EEQ43706.1 | heatshock protein 60, mitochondrial precursor [Candida albicans WO-1] >gi |239613490 | gb | EEQ90477.1 | chaperonin GroL [Ajellomyces dermatitidisER-3] >gi | 240276977 | gb | EER40487.1 | hsp60-like protein[Ajellomyces capsulatus H143] >gi | 241958890 | ref | XP_002422164.1 |heat shock protein 60, mitochondrial precursor, putative [Candidadubliniensis CD36] >gi | 223645509 | emb | CAX40168.1 | heat shockprotein 60, mitochondrial precursor, putative [Candida dubliniensisCD36] >gi | 254572906 | ref | XP_002493562.1 | Tetradecamericmitochondrial chaperonin [Komagataella pastoris GS115] >gi | 238033361 |emb | CAY71383.1 | Tetradecameric mitochondrial chaperonin [Komagataellapastoris GS115] >gi | 254579947 | ref | XP_002495959.1 | ZYRO0C07106p[Zygosaccharomyces rouxii] >gi | 238938850 | emb | CAR27026.1 |ZYRO0C07106p [Zygosaccharomyces rouxii] >gi | 255712781 | ref |XP_002552673.1 | KLTH0C10428p [Lachancea thermotolerans] >gi | 238934052| emb | CAR22235.1 | KLTH0C10428p [Lachancea thermotolerans CBS6340] >gi | 255721795 | ref | XP_002545832.1 | heat shock protein 60,mitochondrial precursor [Candida tropicalis MYA-3404] >gi | 240136321 |gb | EER35874.1 | heat shock protein 60, mitochondrial precursor[Candida tropicalis MYA-3404] >gi | 255941288 | ref | XP_002561413.1 |Pc16g11070 [Penicillium chrysogenum Wisconsin 54-1255] >gi | 211586036 |emb | CAP93777.1 | Pc16g11070 [Penicillium chrysogenum Wisconsin54-1255] >gi | 259148241 | emb | CAY81488.1 | Hsp60p [Saccharomycescerevisiae EC1118] >gi | 260950325 | ref | XP_002619459.1 | heat shockprotein 60, mitochondrial precursor [Clavispora lusitaniae ATCC42720] >gi | 238847031 | gb | EEQ36495.1 | heat shock protein 60,mitochondrial precursor [Clavispora lusitaniae ATCC 42720] >gi |261194577 | ref | XP_002623693.1 | chaperonin GroL [Ajellomycesdermatitidis SLH14081] >gi | 239588231 | gb | EEQ70874.1 | chaperoninGroL [Ajellomyces dermatitidis SLH14081] >gi | 327355067 | gb |EGE83924.1 | chaperonin GroL [Ajellomyces dermatitidis ATCC 18188] >gi |296422271 | ref | XP_002840685.1 | hypothetical protein [Tubermelanosporum Mel28] >gi | 295636906 | emb | CAZ84876.1 | unnamed proteinproduct [Tuber melanosporum] >gi | 296809035 | ref | XP_002844856.1 |heat shock protein 60 [Arthroderma otae CBS 113480] >gi | 238844339 | gb| EEQ34001.1 | heat shock protein 60 [Arthroderma otae CBS 113480] >gi |302308696 | ref | NP_985702.2 | AFR155Wp [Ashbya gossypii ATCC10895] >gi | 299790751 | gb | AAS53526.2 | AFR155Wp [Ashbya gossypiiATCC 10895] >gi | 374108933 | gb | AEY97839.1 | FAFR155Wp [Ashbyagossypii FDAG1] >gi | 302412525 | ref | XP_003004095.1 | heat shockprotein [Verticillium albo-atrum VaMs.102] >gi | 261356671 | gb |EEY19099.1 | heat shock protein [Verticillium albo- atrum VaMs. 102] >gi| 302505585 | ref | XP_003014499.1 | hypothetical protein ARB_07061[Arthroderma benhamiae CBS 112371] >gi | 291178320 | gb | EFE34110.1 |hypothetical protein ARB_07061 [Arthroderma benhamiae CBS 112371] >gi |302656385 | ref | XP_003019946.1 | hypothetical protein TRV_05992[Trichophyton verrucosum HKI 0517] >gi | 291183723 | gb | EFE39322.1 |hypothetical protein TRV_05992 [Trichophyton verrucosum HKI 0517] >gi |302915513 | ref | XP_003051567.1 | predicted protein [Nectriahaematococca mpVI 77-13-4] >gi | 256732506 | gb | EEU45854.1 | predictedprotein [Nectria haematococca mpVI 77-13-4] >gi | 310794550 | gb |EFQ30011.1 | chaperonin GroL [Glomerella graminicola M1.001] >gi |315048491 | ref | XP_003173620.1 | chaperonin GroL [Arthroderma gypseumCBS 118893] >gi | 311341587 | gb | EFR00790.1 | chaperonin GroL[Arthroderma gypseum CBS 118893] >gi | 320580028 | gb | EFW94251.1 |Tetradecameric mitochondrial chaperonin [Ogataea parapolymorphaDL-1] >gi | 320586014 | gb | EFW98693.1 | heat shock proteinmitochondrial precursor [Grosmannia clavigera kw1407] >gi | 322692465 |gb | EFY84374.1 | Heat shock protein 60 precursor (Antigen HIS-62)[Metarhizium acridum CQMa 102] >gi | 322705285 | gb | EFY96872.1 | Heatshock protein 60 (Antigen HIS-62) [Metarhizium anisopliae ARSEF 23] >gi| 323303806 | gb | EGA57589.1 | Hsp60p [Saccharomyces cerevisiaeFostersB] >gi | 323307999 | gb | EGA61254.1 | Hsp60p [Saccharomycescerevisiae FostersO] >gi | 323332364 | gb | EGA73773.1 | Hsp60p[Saccharomyces cerevisiae AWRI796] >gi | 326468648 | gb | EGD92657.1 |heat shock protein 60 [Trichophyton tonsurans CBS 112818] >gi |326479866 | gb | EGE03876.1 | chaperonin GroL [Trichophyton equinum CBS127.97] >gi | 330915493 | ref | XP_003297052.1 | hypothetical proteinPTT_07333 [Pyrenophora teres f. teres 0-1] >gi | 311330479 | gb |EFQ94847.1 | hypothetical protein PTT_07333 [Pyrenophora teres f. teres0-1] >gi | 336271815 | ref | XP_003350665.1 | hypothetical proteinSMAC_02337 [Sordaria macrospora k-hell] >gi | 380094827 | emb |CCC07329.1 | unnamed protein product [Sordaria macrospora k-hell] >gi |336468236 | gb | EGO56399.1 | hypothetical protein NEUTE1DRAFT_122948[Neurospora tetrasperma FGSC 2508] >gi | 340522598 | gb | EGR52831.1 |hsp60 mitochondrial precursor-like protein [Trichoderma reesei QM6a] >gi| 341038907 | gb | EGS23899.1 | mitochondrial heat shock protein 60-likeprotein [Chaetomium thermophilum var. thermophilum DSM 1495] >gi |342886297 | gb | EGU86166.1 | hypothetical protein FOXB_03302 [Fusariumoxysporum Fo5176] >gi | 344230084 | gb | EGV61969.1 | chaperonin GroL[Candida tenuis ATCC 10573] >gi | 344303739 | gb | EGW33988.1 |hypothetical protein SPAPADRAFT_59397 [Spathaspora passalidarum NRRLY-27907] >gi | 345560428 | gb | EGX43553.1 | hypothetical proteinAOL_s00215g289 [Arthrobotrys oligospora ATCC 24927] >gi | 346323592 | gb| EGX93190.1 | heat shock protein 60 (Antigen HIS-62) [Cordycepsmilitaris CM01] >gi | 346975286 | gb | EGY18738.1 | heat shock protein[Verticillium dahliae VdLs.17] >gi | 354545932 | emb | CCE42661.1 |hypothetical protein CPAR2_203040 [Candida parapsilosis] >gi | 358369894| dbj | GAA86507.1 | heat shock protein 60, mitochondrial precursor[Aspergillus kawachii IFO 4308] >gi | 358386867 | gb | EHK24462.1 |hypothetical protein TRIVIDRAFT_79041 [Trichoderma virens Gv29-8] >gi |358399658 | gb | EHK48995.1 | hypothetical protein TRIATDRAFT_297734[Trichoderma atroviride IMI 206040] >gi | 363750488 | ref |XP_003645461.1 | hypothetical protein Ecym_3140 [Eremotheciumcymbalariae DBVPG#7215] >gi | 356889095 | gb | AET38644.1 | Hypotheticalprotein Ecym_3140 [Eremothecium cymbalariae DBVPG#7215] >gi | 365759369| gb | EHN01160.1 | Hsp60p [Saccharomyces cerevisiae × Saccharomyceskudriavzevii VIN7] >gi | 365764091 | gb | EHN05616.1 | Hsp60p[Saccharomyces cerevisiae × Saccharomyces kudriavzevii VIN7] >gi |365985626 | ref | XP_003669645.1 | hypothetical protein NDAI_0D00880[Naumovozyma dairenensis CBS 421] >gi | 343768414 | emb | CCD24402.1 |hypothetical protein NDAI_0D00880 [Naumovozyma dairenensis CBS 421] >gi| 366995970 | ref | XP_003677748.1 | hypothetical protein NCAS_0H00890[Naumovozyma castellii CBS 4309] >gi | 342303618 | emb | CCC71399.1 |hypothetical protein NCAS_0H00890 [Naumovozyma castellii CBS 4309] >gi |367005154 | ref | XP_003687309.1 | hypothetical protein TPHA_0J00520[Tetrapisispora phaffii CBS 4417] >gi | 357525613 | emb | CCE64875.1 |hypothetical protein TPHA_0J00520 [Tetrapisispora phaffii CBS 4417] >gi| 367017005 | ref | XP_003683001.1 | hypothetical protein TDEL_0G04230[Torulaspora delbrueckii] >gi | 359750664 | emb | CCE93790.1 |hypothetical protein TDEL_0G04230 [Torulaspora delbrueckii] >gi |367035486 | ref | XP_003667025.1 | hypothetical protein MYCTH_2097570[Myceliophthora thermophila ATCC 42464] >gi | 347014298 | gb |AEO61780.1 | hypothetical protein MYCTH_2097570 [Myceliophthorathermophila ATCC 42464] >gi | 367055018 | ref | XP_003657887.1 |hypothetical protein THITE_127923 [Thielavia terrestris NRRL 8126] >gi |347005153 | gb | AEO71551.1 | hypothetical protein THITE_127923[Thielavia terrestris NRRL 8126] >gi | 378728414 | gb | EHY54873.1 |heat shock protein 60 [Exophiala dermatitidis NIH/UT8656] >gi |380494593 | emb | CCF33032.1 | heat shock protein 60 [Colletotrichumhigginsianum] >gi | 385305893 | gb | EIF49836.1 | heat shock protein 60[Dekkera bruxellensis AWRI1499] >gi | 389638386 | ref | XP_003716826.1 |heat shock protein 60 [Magnaporthe oryzae 70-15] >gi | 351642645 | gb |EHA50507.1 | heat shock protein 60 [Magnaporthe oryzae 70-15] >gi |440474658 | gb | ELQ43388.1 | heat shock protein 60 [Magnaporthe oryzaeY34] >gi | 440480475 | gb | ELQ61135.1 | heat shock protein 60[Magnaporthe oryzae P131] >gi | 393243142 | gb | EJD50658.1 | chaperoninGroL [Auricularia delicata TFB-10046 SS5] >gi | 396494741 | ref |XP_003844378.1 | similar to heat shock protein 60 [Leptosphaeriamaculans JN3] >gi | 312220958 | emb | CBY00899.1 | similar to heat shockprotein 60 [Leptosphaeria maculans JN3] >gi | 398393428 | ref |XP_003850173.1 | chaperone ATPase HSP60 [Zymoseptoria triticiIPO323] >gi | 339470051 | gb | EGP85149.1 | hypothetical proteinMYCGRDRAFT_75170 [Zymoseptoria tritici IPO323] >gi | 401624479 | gb |EJS42535.1 | hsp60p [Saccharomyces arboricola H-6] >gi | 401842294 | gb| EJT44530.1 | HSP60-like protein [Saccharomyces kudriavzevii IFO1802] >gi | 402076594 | gb | EJT72017.1 | heat shock protein 60[Gaeumannomyces graminis var. tritici R3-111a-1] >gi | 403213867 | emb |CCK68369.1 | hypothetical protein KNAG_0A07160 [Kazachstania naganishiiCBS 8797] >gi | 406606041 | emb | CCH42514.1 | Heat shock protein 60,mitochondrial [Wickerhamomyces ciferrii] >gi | 406863285 | gb |EKD16333.1 | heat shock protein 60 [Marssonina brunnea f. sp.‘multigermtubi’ MB_m1] >gi | 407922985 | gb | EKG16075.1 | ChaperoninCpn60 [Macrophomina phaseolina MS6] >gi | 408399723 | gb | EKJ78816.1 |hypothetical protein FPSE_00959 [Fusarium pseudograminearum CS3096] >gi| 410083028 | ref | XP_003959092.1 | hypothetical protein KAFR_0I01760[Kazachstania africana CBS 2517] >gi | 372465682 | emb | CCF59957.1 |hypothetical protein KAFR_0I01760 [Kazachstania africana CBS 2517] >gi |444315528 | ref | XP_004178421.1 | hypothetical protein TBLA_0B00580[Tetrapisispora blattae CBS 6284] >gi | 387511461 | emb | CCH58902.1 |hypothetical protein TBLA_0B00580 [Tetrapisispora blattae CBS 6284] >gi| 448090588 | ref | XP_004197110.1 | Piso0_004347 [Millerozyma farinosaCBS 7064] >gi | 448095015 | ref | XP_004198141.1 | Piso0_004347[Millerozyma farinosa CBS 7064] >gi | 359378532 | emb | CCE84791.1 |Piso0_004347 [Millerozyma farinosa CBS 7064] >gi | 359379563 | emb |CCE83760.1 | Piso0_004347 [Millerozyma farinosa CBS 7064] >gi |448526196 | ref | XP_003869293.1 | Hsp60 heat shock protein [Candidaorthopsilosis Co 90-125] >gi | 380353646 | emb | CCG23157.1 | Hsp60 heatshock protein [Candida orthopsilosis] >gi | 46123737 | ref | XP_386422.1| HS60_AJECA Heat shock protein 60, mitochondrial precursor (AntigenHIS-62) [Gibberella zeae PH-1] >gi | 50292099 | ref | XP_448482.1 |hypothetical protein [Candida glabrata CBS 138] >gi | 49527794 | emb |CAG61443.1 | unnamed protein product [Candida glabrata] >gi | 50310975 |ref | XP_455510.1 | hypothetical protein [Kluyveromyces lactis NRRLY-1140] >gi | 49644646 | emb | CAG98218.1 | KLLA0F09449p [Kluyveromyceslactis] >gi | 50422027 | ref | XP_459575.1 | DEHA2E05808p [Debaryomyceshansenii CBS767] >gi | 49655243 | emb | CAG87802.1 | DEHA2E05808p[Debaryomyces hansenii CBS767] >gi | 50555023 | ref | XP_504920.1 |YALI0F02805p [Yarrowia lipolytica] >gi | 49650790 | emb | CAG77725.1 |YALI0F02805p [Yarrowia lipolytica CLIB122] >gi | 6323288 | ref |NP_013360.1 | Hsp60p [Saccharomyces cerevisiae S288c] >gi | 123579 | sp| P19882.1 | HSP60_YEAST RecName: Full = Heat shock protein 60,mitochondrial; AltName: Full = CPN60; AltName: Full = P66; AltName: Full= Stimulator factor I 66 kDa component; Flags:Precursor >gi | 171720 |gb | AAA34690.1 | heat shock protein 60 (HSP60) [Saccharomycescerevisiae] >gi | 577181 | gb | AAB67380.1 | Hsp60p: Heat shock protein60 [Saccharomyces cerevisiae] >gi | 151941093 | gb | EDN59473.1 |chaperonin [Saccharomyces cerevisiae YJM789] >gi | 190405319 | gb |EDV08586.1 | chaperonin [Saccharomyces cerevisiae RM11-1a] >gi |207342889 | gb | EDZ70518.1 | YLR259Cp- like protein [Saccharomycescerevisiae AWRI1631] >gi | 256271752 | gb | EEU06789.1 | Hsp60p[Saccharomyces cerevisiae JAY291] >gi | 285813676 | tpg | DAA09572.1 |TPA: chaperone ATPase HSP60 [Saccharomyces cerevisiae S288c] >gi |323353818 | gb | EGA85673.1 | Hsp60p [Saccharomyces cerevisiae VL3] >gi| 349579966 | dbj | GAA25127.1 | K7_Hsp60p [Saccharomyces cerevisiaeKyokai no. 7] >gi | 392297765 | gb | EIW08864.1 | Hsp60p [Saccharomycescerevisiae CEN.PK113-7D] >gi | 226279 | prf | | 1504305A mitochondrialassembly factor >gi | 68485963 | ref | XP_713100.1 | heat shock protein60 [Candida albicans SC5314] >gi | 68486010 | ref | XP_713077.1 | heatshock protein 60 [Candida albicans SC5314] >gi | 6016258 | sp | O74261.1| HSP60_CANAL RecName: Full = Heat shock protein 60, mitochondrial;AltName: Full = 60 kDa chaperonin; AltName: Full = Protein Cpn60; Flags:Precursor >gi | 3552009 | gb | AAC34885.1 | heat shock protein 60[Candida albicans] >gi | 46434552 | gb | EAK93958.1 | heat shock protein60 [Candida albicans SC5314] >gi | 46434577 | gb | EAK93982.1 | heatshock protein 60 [Candida albicans SC5314] >gi | 71001164 | ref |XP_755263.1 | antigenic mitochondrial protein HSP60 [Aspergillusfumigatus Af293] >gi | 66852901 | gb | EAL93225.1 | antigenicmitochondrial protein HSP60, putative [Aspergillus fumigatus Af293] >gi| 159129345 | gb | EDP54459.1 | antigenic mitochondrial protein HSP60,putative [Aspergillus fumigatus A1163] >gi | 90970323 | gb | ABE02805.1| heat shock protein 60 [Rhizophagus intraradices]

In an embodiment, a 10 kDa chaperone from Table 3 is combined with amatching 60 kDa chaperone from table 4 of the same organism genus orspecies for expression in the host.

For instance: >gi|189189366|ref|XP_(—)001931022.1|:71-168 10 kDachaperonin [Pyrenophora tritici-repentis] expressed together withmatching>gi|189190432|ref|XP_(—)001931555.1| heat shock protein 60,mitochondrial precursor [Pyrenophora tritici-repentis Pt-1C-BFP].

All other combinations from Table 3 and 4 similarly made with sameorganism source are also available to the skilled person for expression.

Further, one may combine a chaperone from Table 3 from one organism witha chaperone from Table 4 from another organism, or one may combine GroESwith a chaperone from Table 3, or one may combine GroEL with a chaperonefrom Table 4.

As follows from the above, the invention further relates to a method forpreparing an organic compound comprising converting a carbon source,using a microorganism, thereby forming the organic compound. The methodmay be carried out under aerobic, oxygen-limited or anaerobicconditions.

The invention allows in particular a reduction in formation of an NADHdependent side-product, especially glycerol, by up to 100%, up to 99%,or up to 90%, compared to said production in a corresponding referencestrain. The NADH dependent side-product formation is preferably reducedby more than 10% compared to the corresponding reference strain, inparticular by at least 20%, more in particular by at least 50%. NADHdependent side-product production is preferably reduced by 10-100%, inparticular by 20-95%, more in particular by 50-90%.

In preferred method wherein Rubisco, or another enzyme capable ofcatalysing the formation of an organic compound from CO₂ (and anothersubstrate) or another enzyme that catalyses the function of CO₂ as anelectron acceptor, is used, the carbon dioxide concentration in thereaction medium is at least 5% of the CO₂ saturation concentration underthe reaction conditions, in particular at least 10% of said CO₂saturation concentration, more in particular at least 20% of said CO₂saturation concentration. This is in particular advantageous withrespect to product yield. The reaction medium may be oversaturated inCO₂ concentration, saturated in CO₂ concentration or may have aconcentration below saturation concentration. In a specific embodiment,the CO₂ concentration is 75% of the saturation concentration or less, inparticular 50% of said saturation concentration or less, more inparticular is 25% of the CO₂ saturation concentration or less.

In a specific embodiment, the carbon dioxide or part thereof is formedin situ by the microorganism. If desired, the method further comprisesthe step of adding external CO₂ to the reaction system, usually byaeration with CO₂ or a gas mixture containing CO₂, for instance aCO₂/nitrogen mixture. Adding external CO₂ in particular is used to(increase or) maintain the CO₂ within a desired concentration range, ifno or insufficient CO₂ is formed in situ.

Determination of the CO₂ concentration in a fluid is within the routineskills of the person skilled in the art. In practice, one may routinelydetermine the CO₂ concentration in the gas phase above a culture of theyeast (practically the off-gas if the medium is purged with a gas). Thiscan routinely be measured using a commercial gas analyser, such as aRosemountNGA200000 gas analyser (Rosemount Analytical, Orrvile, USA).The concentration in the liquid phase (relative to the saturationconcentration), can then be calculated from the measured value in thegas, from the CO₂ saturation concentration and Henri coefficients ofunder the existing conditions in the method. These parameters areavailable from handbooks or can be routinely determined.

As a carbon source, in principle any carbon source that themicroorganism can use as a substrate can be used. In particular anorganic carbon source may be used, selected from the group ofcarbohydrates and lipids (including fatty acids). Suitable carbohydratesinclude monosaccharides, disaccharides, and hydrolysed polysaccharides(e.g. hydrolysed starches, lignocellulosic hydrolysates). Although acarboxylic acid may be present, it is not necessary to include acarboxylic acid such as acetic acid, as a carbon source.

It is in particular an advantage of the present invention that animproved ethanol yield and a reduced glycerol production is feasiblecompared to, e.g., a wild type yeast cell, without needing to intervenein the genome of the cell by inhibition of a glycerol 3-phosphatephosphohydrolase and/or encoding a glycerol 3-phosphate dehydrogenasegene.

Still, in a specific embodiment, a yeast cell according to the inventionmay comprise a deletion or disruption of one or more endogenousnucleotide sequence encoding a glycerol 3-phosphate phosphohydrolaseand/or encoding a glycerol 3-phosphate dehydrogenase gene:

Herein in the cell, enzymatic activity needed for the NADH-dependentglycerol synthesis is reduced or deleted. The reduction or deleted ofthis enzymatic activity can be achieved by modifying one or more genesencoding a NAD-dependent glycerol 3-phosphate dehydrogenase activity(GPD) or one or more genes encoding a glycerol phosphate phosphataseactivity (GPP), such that the enzyme is expressed considerably less thanin the wild-type or such that the gene encoded a polypeptide withreduced activity.

Such modifications can be carried out using commonly knownbiotechnological techniques, and may in particular include one or moreknock-out mutations or site-directed mutagenesis of promoter regions orcoding regions of the structural genes encoding GPD and/or GPP.Alternatively, yeast strains that are defective in glycerol productionmay be obtained by random mutagenesis followed by selection of strainswith reduced or absent activity of GPD and/or GPP. S. cerevisiae GPD1,GPD2, GPP1 and GPP2 genes are shown in WO 2011/010923, and are disclosedin SEQ ID NO: 24-27 of that application. The contents of thisapplication are incorporated by reference, in particular the contentsrelating to GPD and/or GPP.

As shown in the Examples below, the invention is in particular found tobe advantageous in a process for the production of an alcohol, notablyethanol. However, it is contemplated that the insight that CO₂ can beused as an electron acceptor in microorganisms that do not naturallyallow this, has an industrial benefit for other biotechnologicalprocesses for the production of organic molecules, in particular organicmolecules of a relatively low molecular weight, particularly organicmolecules with a molecular weight below 1000 g/mol. The following itemsare mentioned herein as preferred embodiments of the use of carbondioxide as an electron acceptor in accordance with the invention.

1. Use of carbon dioxide as an electron acceptor in a recombinantchemotrophic micro-organism is a non-phototrophic eukaryoticmicro-organism.

2. Use of carbon dioxide as an electron acceptor in a recombinantchemotrophic micro-organism, wherein the micro-organism produces anorganic compound under anaerobic conditions.

3. Use according to item 1 or 2, wherein the carbon dioxide serves as anelectron acceptor in a process with NADH as an electron donor.

5. Use according to any of the preceding items, wherein themicro-organism produces an organic compound in a process with an excessproduction of ATP and/or NADH.

6. Use according to any of the preceding items, wherein themicro-organism comprises a heterologous nucleic acid sequence encoding apolypeptide from a (naturally) autotrophic organism.

7. Use according to item 6, wherein the micro-organism comprises aheterologous nucleic acid sequence encoding a first prokaryoticchaperone for said polypeptide and preferably a nucleic acid sequenceencoding a second prokaryotic chaperone—different from the first—forsaid polypeptide.

8. Use according to item 7, wherein the chaperones are GroEL and GroES.

9. Use according to any of the preceding items, wherein themicro-organism produces an organic compound selected from the groupconsisting of alcohols (such as methanol, ethanol, propanol, butanol,phenol, polyphenol), ribosomal peptides, antibiotics (such aspenicillin), bio-diesel, alkynes, alkenes, isoprenoids, esters,carboxylic acids (such as succinic acid, citric acid, adipic acid,lactic acid), amino acids, polyketides, lipids, and carbohydrates.

10. Use according to any of the preceding items, wherein themicroorganism comprises a heterologous nucleic acid sequencefunctionally expressing a polypeptide selected from the group consistingof carbonic anhydrases, carboxylases, oxygenases, hydrogenases,dehydrogenases, isomerases, aldolases, transketolases, transaldolases,phosphatases, epimerases, kinases, carboxykinases, oxidoreductases,aconitases, fumarases, reductases, lactonases, phosphoenolpyruvate (PEP)carboxylases, phosphoglycerate kinases, glyceraldehyde 3-phosphatedehydrogenases, triose phosphate isomerases,fructose-1,6-bisphosphatases, sedoheptulose-1,7-bisphosphatases,phosphopentose isomerases, phosphopentose epimerase,phosphoribulokinases (PRK), glucose 6-phosphate dehydrogenases,6-phosphogluconolactonases, 6-phosphogluconate dehydrogenases, ribulose5-phosphate isomerases, ribulose 5-phosphate 3-epimerases,Ribulose-1,5-bisphosphate carboxylase oxygenases, lactatedehydrogenases, malate synthases, isocitrate lyases, pyruvatecarboxylases, phosphoenolpyruvate carboxykinases,fructose-1,6-bisphosphatases, phosphoglucoisomerases,glucose-6-phosphatases, hexokinases, glucokinases, phosphofructokinases,pyruvate kinases, succinate dehydrogenases, citrate synthases,isocitrate dehydrogenases, α-ketoglutarate dehydrogenases, succinyl-CoAsynthetases, malate dehydrogenases, nucleoside-diphosphate kinases,xylose reductases, xylitol dehydrogenases, xylose isomerases, isoprenoidsynthases, and xylonate dehydratases.

11. Use according to item 10, wherein the microorganism comprises aheterologous nucleic acid sequence functionally expressingRibulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) and/or aheterologous nucleic acid sequence functionally expressing aphosphoribulokinase (PRK).

12. Use according to any of the preceding items, wherein themicroorganism is selected from the group of is selected from the groupconsisting of Saccharomyceraceae, Penicillium, Yarrowia and Aspergillus.

13. Use according to any of the preceding items, wherein the carbondioxide is used as an electron acceptor to reduce production of anNAD+-dependent side-product or NADH-dependent side-product, such asglycerol, in a process for preparing another organic compound, such asanother alcohol or a carboxylic acid.

14. Recombinant micro-organism, in particular a eukaryoticmicro-organism, having an enzymatic system allowing the micro-organismto use carbon dioxide as an electron acceptor under chemotrophic(non-phototrophic) conditions, wherein the microorganism is preferablyas defined in the prevision items.

15. Recombinant micro-organism according to item 14, wherein themicro-organism has an enzymatic system for producing an organic compoundin a process with an excess production of ATP and/or NADH.

The production of the organic compound of interest may take place in aorganism known for it usefulness in the production of the organiccompound of interest, with the proviso that the organism has beengenetically modified to enable the use of carbon dioxide as an electronacceptor in the organism.

Although it is contemplated that the invention is interesting for theproduction of a variety of industrially relevant organic compounds, amethod or use according the invention is in particular consideredadvantageous for the production of an alcohol, in particular an alcoholselected from the group of ethanol, n-butanol and 2,3-butanediol; or inthe production of an organic acid/carboxylate, in particular acarboxylate selected from the group of L-lactate, 3-hydroxypropionate,D-malate, L-malate, succinate, citrate, pyruvate and itaconate.

Regarding the production of ethanol, details are found herein above,when describing the yeast cell comprising PRK and Rubisco and in theexamples. The ethanol or another alcohol is preferably produced in afermentative process.

For the production of several organic acids (carboxylates), e.g. citricacid, an aerobic process is useful. For citric acid production forinstance Aspergillus niger, Yarrowia lipolytica, or another knowncitrate producing organism may be used.

An example of an organic acid that is preferably produced anaerobicallyis lactic acid. Various lactic acid producing bacterial strains andyeast strains that have been engineered for lactate production aregenerally known in the art.

EXAMPLES Example 1 Construction of the Expression Vector

Phosphoribulokinase (PRK) cDNA from Spinacia oleracea (spinach) (EMBLaccession number: X07654.1) was PCR-amplified using Phusion Hot-startpolymerase (Finnzymes, Landsmeer, the Netherlands) and theoligonucleotides XbaI_prk-FW2 and RV1_XhoI_prk (Table 5), and wasligated in pCR®-Blunt II-TOPO® (Life Technologies Europe BV, Bleiswijk,the Netherlands).

TABLE 5 Oligonucleotides Sequence Number Name (5′ to 3′) Purpose Cloning 1 XbaI_prk_FW2 TGACATCTAGATGTCACAA cloning of PRK into pUDE046.CAACAAACAATTG  2 RV1 XhoI prk TGACATCTAGATGTCACAAcloning of PRK into pUDE046. CAACAAACAATTGPrimers used for in vivo plasmid assembly  3 HR-cbbM-FW-65TTGTAAAACGACGGCCAGT Rubisco cbbM cassette for plasmidsGAGCGCGCGTAATACGACT pUDC075, pUDC099, and pUDC100. CACTATAGGGCGAATTGGGTACAGCTGGAGCTCAGTTT ATCATTATC  4 HR-cbbM-RV-65 GGAATCTGTGTAGTATGCCRubisco cbbM cassette for plasmids TGGAATGTCTGCCGTGCCApUDC075, pUDC099, and pUDC100 TAGCCATGTATGCTGATAT GTCGGTACCGGCCGCAAATTAAAG  5 linker-cbbO2-pRS416 ATCACTCTTACCAGGCTAGLinker fragment for assembly of plasmid GACGACCCTACTCATGTAT pUDC099.TGAGATCGACGAGATTTCT AGGCCAGCTTTTGTTCCCT TTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGT CATAGC  6 linker-cbbM-GroEL GACATATCAGCATACATGGLinker fragment for assembly of plasmid CTATGGCACGGCAGACATT pUDC100.CCAGGCATACTACACAGAT TCCATCACTCTTACCAGGC TAGGACGACCCTACTCATGTATTGAGATCGACGAGATT TCTAGG Primers used for in vivo integration assembly 7 FW pTDH3-HR-CAN1up GTTGGATCCAGTTTTTAAT1^(st) cloning expression cassette linker CTGTCGTCAATCGAAAGTTfragment between CAN1 upstream and TATTTCAGAGTTCTTCAGAPRK expression cassette (IMI229), and CTTCTTAACTCCTGTAAAACAN1up-linker and KlLEU2 expression ACAAAAAAAAAAAAAGGCAcassette (IMI232). TAGCAAGCTGGAGCTCAGT TTATC  8 RV linker-iHR2BAGATATACTGCAAAGTCCG 1^(st) cloning fragment: linker fragmentGAGCAACAGTCGTATAACT between CAN1up-linker and PRK CGAGCAGCCCTCTACTTTGexpression cassette (IMI229). TTGTTGCGCTAAGAGAATG GACC  9 RV linker-iHR6GCTATGACCATGATTACGC 1^(st) cloning fragment: linker fragmentCAAGCGCGCAATTAACCCT between CAN1up-linker and KlLEU2 CACTAAAGGGAACAAAAGCexpression cassette (IMI232). TGGTTGCGCTAAGAGAATG GACC 10FW pGAL1-prk HR2B CAACAAAGTAGAGGGCTGC2^(nd) cloning fragment: GAL1_(p)-PRK-CYC1_(t) TCGAGTTATACGACTGTTGexpression cassette (IMI229) from CTCCGGACTTTGCAGTATA pUDE046.TCTGCTGGAGCTCTAGTAC GGATT 11 RV CYC1t-prk HR2 GGAATCTGTGTAGTATGCC2^(nd) cloning fragment: GAL1p-PRK-CYC1_(t) TGGAATGTCTGCCGTGCCAexpression cassette (IMI229) from TAGCCATGTATGCTGATAT pUDE046.GTCGTACCGGCCGCAAATT AAAG 12 FW HR2-cbbQ2-HR3 GACATATCAGCATACATGG3^(rd)I cloning fragment: PGI1_(p)-cbbQ2- CTATGGTEF2t cassette (IMI229). 13 RV HR2-cbbQ2-HR3 GGACACGCTTGACAGAATG3^(rd) cloning fragment: PGI1_(p)-cbbQ2- TCAAAGGTEF2t cassette (IMI229). 14 FW HR3-cbbO2-HR4 CGTCCGATATGATCTGATT4^(th) TARI cloning fragment: PGK1_(p)- GGcbbO2-ADH1_(t) cassette (IMI229). 15 RV HR3-cbbO2-HR4CCTAGAAATCTCGTCGATC 4^(th) cloning fragment: PGK1_(p)-cbbO2- TCADH1_(t) cassette (IMI229). 16 FW HR4-GroEL-HR5 ATCACTCTTACCAGGCTAG5^(th) cloning fragment:TEF1_(p)-groEL-ACT1_(t) G cassette (IMI229). 17RV HR4-GroEL-HR5 CTGGACCTTAATCGTGTGC5^(th) cloning fragment: TEF1_(p)-groEL- GCATCCTCACT1_(t) cassette (IMI229). 18 FW HR5-GroES-HR6 CCGTATAGCTTAATAGCCA6^(th) cloning fragment: TPI1_(p)-groES-PGI1_(t) GCTTTATCcassette (IMI229). 19 RV HR5-GroES-HR6 GCTATGACCATGATTACGC6^(th) cloning fragment: TPI1_(p)-groES-PGI1_(t) CAAGCcassette (IMI229). 20 FW HR6-LEU2-CAN1dwn CCAGCTTTTGTTCCCTTTA7^(th) (IMI229) or 2nd (IMI232) cloning GTGAGGGTTAATTGCGCGCfragment: KlLEU2 cassette from pUG73. TTGGCGTAATCATGGTCATAGCCTGTGAAGATCCCAGC AAAG 21 RV LEU2 HR-CAN1 AGCTCATTGATCCCTTAAA7^(th) (IMI229) or 2nd (IMI232) cloning CTTTCTTTTCGGTGTATGAfragment: KlLEU2 cassette from pUG73. CTTATGAGGGTGAGAATGCGAAATGGCGTGGAAATGTG ATCAAAGGTAATAAAACGT CATATATCCGCAGGCTAAC CGGAACPrimers used for verification of the in vivo assembled constructs 22m-PCR-HR1-FW GGCGATTAAGTTGGGTAAC Diagnostic for assembly of plasmids GpUDC075, pUDC099, and pUDC100,. 23 m-PCR-HR1-RV AACTGAGCTCCAGCTGTACDiagnostic for assembly of plasmids C pUDC075, pUDC099, pUDC100, andintegration in strain IMI229. 24 m-PCR-HR2-FW ACGCGTGTACGCATGTAACDiagnostic for assembly of pUDC075, pUDC099, pUDC100, and integration instrain IMI229 25 m-PCR-HR2-RV GCGCGTGGCTTCCTATAATDiagnostic for assembly of pUDC075, CpUDC099, pUDC100, and integration in strain IMI229 26 m-PCR-HR3-FWGTGAATGCTGGTCGCTATA Diagnostic for assembly of pUDC075, CpUDC099, pUDC100, and integration in strain IMI229. 27 m-PCR-HR3-RVGTAAGCAGCAACACCTTCA Diagnostic for assembly of pUDC075, GpUDC099, pUDC100, and integration in strain IMI229. 28 m-PCR-HR4-FWACCTGACCTACAGGAAAGA Diagnostic for assembly of pUDC075, GpUDC099, pUDC100, and integration in strain IMI229. 29 m-PCR-HR4-RVTGAAGTGGTACGGCGATGC Diagnostic for assembly of pUDC075,pUDC099, pUDC100, and integration in strain IMI229. 30 m-PCR-HR5-FWATAGCCACCCAAGGCATTT Diagnostic for assembly of pUDC075, CpUDC099, pUDC100, and integration in strain IMI229. 31 m-PCR-HR5-RVCCGCACTTTCTCCATGAGG Diagnostic for assembly of pUDC075,pUDC099, pUDC100, and integration in strain IMI229. 32 m-PCR-HR6-FWCGACGGTTACGGTGTTAAG Diagnostic for assembly of pUDC075,pUDC099, pUDC100, and integration in strain IMI229. 33 m-PCR-HR6-RVCTTCCGGCTCCTATGTTGT Diagnostic for assembly of pUDC075, GpUDC099, pUDC100, and integration in strain IMI229.

After restriction by XbaI and XhoI, the PRK-containing fragment wasligated into pTEF424. The TEF1p was later replaced by GAL1p from plasmidpSH47 by XbaI and SacI restriction/ligation, creating plasmid pUDE046(see Table 6).

TABLE 6 Plasmids Name Relevant genotype Source/reference pFL451pAOX1-prk (Spinach)-AOX1t (pHIL2-D2 HIS4 Amp Brandes et al. centromeric)1996.¹⁴ pCR ®-Blunt bla Life II-TOPO Technologies Europe BV pTEF424_TEFTRP1 2μ bla Mumberg et al. 1995²⁵. pSH47 URA3 CEN6 ARS4GAL1_(p)-cre-CYC1_(t) bla Güldener et al 1996²⁶ pUD0E46 TRP1 2μGAL1p-prk-CYC1_(t) bla This study. pPCR-Script bla Life TechnologiesEurope BV pGPD_426 URA3 2μ bla Mumberg et al. 1995²⁵. pRS416 URA3 CEN6ARS4 bla Mumberg et al. 1995²⁵. pBTWW002 URA3 2μ TDH3_(p)-cbbM-CYC1_(t)bla This study. pUDC098 URA3 CEN6 ARS4 TDH3_(p)-cbbM-CYC1_(t) bla Thisstudy. pMK-RQ nptII Life Technologies Europe BV pUD230PGI1_(p)-cbbQ2-TEF2_(t) nptII Life Technologies Europe BV pUD231PGK1_(p)-cbbO2-ADH1_(t) nptII Life Technologies Europe BV pUD232TEF1_(p)-groEL-ACT1_(t) nptII Life Technologies Europe BV pUD233TPI1_(p)-groES-PGI1_(t) nptII Life Technologies Europe BV pUDC075 URA3CEN6 ARS4 TDH3_(p)-cbbM-CYC1_(t;)PGI1_(p)-cbbQ2- This study.TEF2_(t);PGK1_(p)-cbbO2-ADH1_(t);TEF1_(p)-groEL-ACT1_(t);TPI1_(p)-groES-PGI1_(t) bla pUDC099 URA3 CEN6 ARS4TDH3_(p)-cbbM-CYC1_(t;)PGI1_(p)-cbbQ2- This study.TEF2_(t);PGK1_(p)-cbbO2-ADH1_(t) bla pUDC100 URA3 CEN6 ARS4TDH3_(p)-cbbM-CYC1_(t;) TEF1_(p)-groEL- This study.ACT1_(t);TPI1_(p)-groES-PGI1_(t) bla

Rubisco form II gene cbbM from Thiobacillus denitrificans (T.denitrificans) flanked by KpnI and SacI sites was codon optimizedsynthesized at GeneArt (Life Technologies Europe BV), and ligated intopPCR-Script, the plasmid was then digested by BamHI and SacI. ThecbbM-containing fragment was ligated into the BamHI and SacI restrictedvector pGPD_(—)426 creating plasmid pBTWW002. The cbbM expressioncassette was transferred into pRS416 using KpnI and SacI, yieldingpUDC098.

Expression cassette of the specific Rubisco form II cheparones from T.denitrificans cbbQ2 and cbbO2, and chaperones groEL and groES from E.coli. were condon optimized. The expression cassettes contained a yeastconstitutive promoters and terminator, flanking the codon optimizedgene. The cassette was flanked by unique 60 bp regions obtained byrandomly combining bar-code sequences used in the Saccharomyces GenomeDeletion Project and an EcoRV site (GeneArt). The expression cassetteswere inserted in plasmid pMK-RQ (GeneArt) using the SfiI cloning sitesyielding pUB230 (PGI1p-cbbQ2-TEF2t), pUD231 (PGK1p-cbbO2-ADH1t), pUD232(TEF1p-groEL-ACT1t), and pUDE233 (TPI1p-groES-PGI1t) Table 6). Theexpression cassette TDH3p-cbbM-CYC1t was PCR-amplified from plasmidpBTWW002 using Phusion Hot-Start Polymerase (Finnzymes) and primersHR-cbbM-FW-65 and HR-cbbM-RV-65 in order to incorporate the 60-bp regionfor recombination cloning.

Example 2 Strain Construction, Isolation and Maintenance

All Saccharomyces cerevisiae strains used (Table 7) belong to the CEN.PKfamily. All strains were grown in 2% w/v glucose synthetic mediasupplemented with 150 mg L⁻¹ uracil when required until they reached endexponential phase, then sterile glycerol was added up to ca. 30% v/v andaliquots of 1 ml were stored at −80° C.

TABLE 7 Saccharomyces cerevisiae strains Strain Relevant genotypeSource/reference CEN.PK113-5D MATa ura3-52 Euroscarf CEN.PK102-3A MATaura3-52 leu2-3, 112 Euroscarf IMC014 MATa ura3-52 pUDC075 (CEN6 ARS4URA3 TDH3_(P)- This study. cbbM-CYC1_(t) PGI1_(p)-cbbQ2-TEF2_(t)PGK1_(p)-cbbO2- ADH1_(t) TEF1_(p)-groEL-ACT1_(t)TPI1_(p)-groES-PGI1_(t)) IMC033 MATa ura3-52 pUDC098 (CEN6 ARS4 URA3TDH3_(p)- This study. cbbM-CYC1_(t)) IMC034 MATa ura3-52 pUDC099 (CEN6ARS4 URA3 TDH3_(p)- This study. cbbM-CYC1_(t) PGI1_(p)-cbbQ2-TEF2_(t)PGK1_(p)-cbbO2- ADH1_(t)cbbO2-pRS416 linker) IMC035 MATa ura3-52 pUDC100(CEN6 ARS4 URA3 TEF1_(P)- This study. groEL-ACT1_(t)TPI1_(p)-groES-PGI1_(t) cbbM-GroEL linker) IMI229 MATa ura3-52 leu2-3,112 can1Δ::GAL1_(p)-prk-CYC1_(t) This study.PGI1_(p)-cbbQ2-TEF2_(t),PGK1_(p)-cbbO2-ADH1_(t),TEF1_(p)-groEL-ACT1_(t),TPI1_(p)-groES-PGI1_(t) KlLEU2 IMI232 MATa ura3-52leu2-3, 112 can1Δ::KlLEU2 This study. IMU032 IMI232 p426_GPD (2μ URA3)This study. IMU033 IMI229 pUDC100 (CEN6 ARS4 URA3 TEF1_(p)-groEL- Thisstudy. ACT1_(t) TPI1_(p)-groES-PGI1_(t) cbbM-GroEL linker)

The strain IMC014 that co-expressed the Rubisco form II ccbM and thefour chaperones cbbQ2, ebbO2, groEL, and groES was constructed using invivo transformation associated recombination. 200 fmol of eachexpression cassette were pooled with 100 fmol of the KpnI/SacIlinearized pRS416 backbone in a final volume of 500 and transformed inCEN.PK 113-5D using the lithium acetate protocol (Gietz, et al., YeastTransformation by the LiAc/SS Carrier DNA/PEG Method in Yeast Protocol,Humana press, 2006). Cells were selected on synthetic medium. Correctassembly of the fragment of pUDC075 was performed by multiplex PCR ontransformant colonies using primers enabling amplification over theregions used for homologous recombination (Table 5) and by restrictionanalysis after re-transformation of the isolated plasmid in E. coliDH5α. PUDC075 was sequenced by Next-Generation Sequencing (Illumina, SanDiego, Calif., U.S.A.) (100br reads paired-end, 50 Mb) and assembledwith Velvet (Zerbino, et al., Velvet: Algorithms for De Novo Short ReadAssembly Using De Bruijn Graphs, Genome Research, 2008). The assembledsequence did not contain mutations in any of the assembled expressioncassettes. The strains IMC034 and IMC035 that expressed ccbM/ccbQ2/ccbO2and ccbM/groEL/groES respectively were constructed using the same invivo assembly method with the following modification. To constructplasmids pUDC099 and pUDC100, 120 bp cbbO2-pRS416 linker and cbbM-GroELlinker were used to close the assembly respectively (Table 5), 100 fmolof each of complementary 120 bp oligonucleotides were added to thetransformation. The strain IMC033 that only expressed the cbbM gene wasconstructed by transforming CEN.PK113-5D with pUDC098.

To construct the strain IMU033 that co-expressed PRK, ccbM, ccbQ2,ccbO2, GroEL, GroES, the intermediate strain IM1229 was constructed byintegrating PRK, the four chaperones and KlLEU2 (Güldener, et al., Asecond set of loxP marker cassettes for Cre-mediated multiple geneknockouts in budding yeast, Nucleic Acids Research, 2002) at the CAN1locus by in vivo homologous integration in CEN.PK102-3A. The expressioncassettes were PCR amplified using Phusion Hot-Start Polymerase(Finnzymes, Thermo Fisher Scientific Inc. Massachusetts, U.S.A.), thecorresponding oligonucleotides and DNA templates (Table 5). Finally, thestrain IM1229 was transformed with pUDC100 that carries the Rubisco formII ccbM and the two E. coli chaperones groEL and groES.

Strain IM1232 was constructed by transforming CEN.PK102-3A with theKlLEU2 cassette. IM1232 was finally transformed with the plasmid p426GPDto restore prototrophy resulting in the reference strain IMU032.

Example 3 Experimental Set-Up of Chemostat and Batch Experiments

Anaerobic chemostat cultivation was performed essentially as described(Basso, et al., Engineering topology and kinetics of sucrose metabolismin Saccharomyces cerevisiae for improved ethanol yield, MetabolicEngineering 13:694-703, 2011), but with 12.5 g 1-1 glucose and 12.5 g1-1 galactose as the carbon source and where indicated, a mixture of 10%CO₂/90% N₂ replaced pure nitrogen as the sparging gas. Residual glucoseand galactose concentrations were determined after rapid quenching(Mashego, et al., Critical evaluation of sampling techniques forresidual glucose determination in carbon-limited chemostat culture ofSaccharomyces cerevisiae, Biotechnology and Bioengineering 83:395-399,2003) using commercial enzymatic assays for glucose (Boehringer,Mannheim, Germany) and D-galactose (Megazyme, Bray, Ireland). Anaerobicbioreactor batch cultures were grown essentially as described (GuadalupeMedina, et al., Elimination of glycerol production in anaerobic culturesof a Saccharomyces cerevisiae strain engineered to use acetic acid as anelectron acceptor. Applied and Environmental Microbiology 76:190-195,2010), but with 20 g L⁻¹ galactose and a sparging gas consisting of 10%CO₂ and 90% N₂. Biomass and metabolite concentrations in batch andchemostat and batch cultures were determined as described by Guadalupeet al. (Guadalupe Medina, et al., Elimination of glycerol production inanaerobic cultures of a Saccharomyces cerevisiae strain engineered touse acetic acid as an electron acceptor. Appl. Environ. Microbiol. 76,190-195, 2010). In calculations of ethanol fluxes and yields, ethanolevaporation was corrected for based on a first-order evaporation rateconstant of 0.008 h⁻¹ in the bioreactor set-ups and under the conditionsused in this study.

Example 4 Enzyme Assays for Phosphoribulokinase (PRK) and Rubisco

Cell extracts for analysis of phosphoribulokinase (PRK) activity wereprepared as described previously (Abbott, et al., CatalaseOverexpression reduces lactic acid-induced oxidative stress inSaccharomyces cerevisiae, Applied and Environmental Microbiology75:2320-2325, 2009). PRK activity was measured at 30° C. by a coupledspectrophotometric assay (MacElroy, et al., Properties ofPhosphoribulokinase from Thiobacillus neapolitanus, Journal ofBacteriology 112:532-538, 1972). Reaction rates were proportional to theamounts of cell extract added. Protein concentrations were determined bythe Lowry method (Lowry, et al., Protein measurement with the Folinphenol reagent, The Journal of Biological Chemistry 193:265-275, 1951)using bovine serum albumin as a standard.

Cell extracts for Rubisco activity assays were prepared as described inAbbott, D. A. et al. Catalase overexpression reduces lactic acid-inducedoxidative stress in Saccharomyces cerevisiae. Appl. Environ. Microbiol.75:2320-2325, 2009, with two modifications: Tris-HCl (1 mM, pH 8.2)containing 20 mM MgCl₂.6H₂O, 5 mM of DTT 5 mM NaHCO₃ was used assonication buffer and Tris-HCl (100 mM, pH 8.2), 20 mM MgCl₂.6H₂O and 5mM of DTT as freezing buffer. Rubisco activity was determined bymeasuring ¹⁴CO₂-fixation (PerkinElmer, Groningen, The Netherlands) asdescribed (Beudeker, et al., Relations betweend-ribulose-1,5-biphosphate carboxylase, carboxysomes and CO₂ fixingcapacity in the obligate chemolithotroph Thiobacillus neapolitanus grownunder different limitations in the chemostat, Archives of Microbiology124:185-189, 1980) and measuring radioactive counts in a TRI-CARBO2700TR Series liquid scintillation counter (PerkinElmer, Groningen, TheNetherlands), using Ultima Gold™ scintillation cocktail (PerkinElmer,Groningen, The Netherlands). Protein concentrations were determined bythe Lowry method (Lowry, O. H., Rosebrough, N. J., Farr, A. L., &Randall, R. J. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193:265-275, 1951) using standard solutions of bovine serumalbumin dissolved in 50 mM Tris-HCl (pH 8.2).

Example 5 The Activity of Rubisco and the Activity of PRK in CellExtracts

In order to study a possible requirement of heterologous chaperones ofRubisco in S. cerevisiae, the form-II Rubisco-encoding cbbM gene from T.denitrificans was codon-optimised and expressed from a centromericvector, both alone and in combination with expression cassettes for thecodon-optimised E. coli groEL/groES and/or T. denitrificans cbbO2/cbbQ2genes. Analysis of ribulose-1,5-biphosphate-dependent CO₂ fixation byyeast cell extracts demonstrated that functional expression of T.denitrificans Rubisco in S. cerevisiae was observed upon co-expressionof E. coli GroEL/GroES. Rubisco activity increased from <0.2 nmolmin⁻¹.(mg protein)⁻¹ to more than 6 nmol min⁻¹.(mg protein)⁻¹. Resultsof these experiments are visualised in FIG. 1, showing specificribulose-1,5-bisphosphate carboxylase (Rubisco) activity in cellextracts of S. cerevisiae expressing Rubisco form II CbbM from T.denitrificans either alone (IMC033) or in combination with the E. colichaperones GroEL/GroES (IMC035), The T. denitrificans chaperonesCbbO2/CbbQ2 [20] (IMC034) or all four chaperones (IMC014).Heterologously expressed genes were codon optimised for expression inyeast and expressed from a single centromeric vector. Biomass sampleswere taken from anaerobic batch cultures on synthetic media (pH 5.0, 30°C.), sparged with nitrogen and containing 20 g 1-1 glucose as carbonsource. Rubisco activities, measured as 14CO2-fixation in cell extracts,in a wild-type reference strain and in S. cerevisiae strains expressingcbbM and cbbM-cbbQ2-cbbO2 were below the detection limit of the enzymeassay (0.2 nmol CO2 min-1 mg protein-1

Co-expression of CbbO2/cbbQ2 did not result in a significant furtherincrease of Rubisco activity. The positive effect of GroEL/GroES onRubisco expression in S. cerevisiae demonstrates the potential value ofthis approach for metabolic engineering, especially when prokaryoticenzymes need to be functionally expressed in the cytosol of eukaryotes.

The Spinach oleracea PRK gene was integrated together with E. coligroEL/groES and T. denitrificans cbbO2/cbbQ2 into the S. cerevisiaegenome at the CAN1 locus, under control of the galactose-inducible GAL1promoter. This induced in high PRK activities in cell extracts of S.cerevisiae strain IMU033, which additionally carried the centromericexpression cassette for T. denitrificans Rubisco. This engineered yeaststrain was used to quantitatively analyze the physiological impacts ofthe expression of Rubisco and PRK.

TABLE 8 IMU032 IMU033 (expressing (reference strain) PRK and Rubisco)CO₂ in inlet gas (%) 0 10 0 10 CO₂ in outlet gas (%) 0.89 ± 10.8 ± 1.02± 10.8 ± 0.03 0.0 0.00 0.1 Phosphoribulokinase 0.58 ± 0.51 ± 14.4 ± 15.2± (μmol mg protein⁻¹ 0.09 0.12 1.5 1.0 min⁻¹) Rubisco (nmol mg <0.2*<0.2 4.59 ± 2.67 ± protein⁻¹ min⁻¹) 0.30 0.28 Biomass yield on sugar0.083 ± 0.084 ± 0.093 ± 0.095 ± (g g⁻¹) 0.000^(a) 0.000^(b) 0.001^(a)0.000^(b) Ethanol yield on sugar 1.56 ± 1.56 ± 1.73 ± 1.73 ± (mol mol⁻¹)0.03^(c) 0.02^(d) 0.02^(c) 0.01^(d) Glycerol yield on sugar 0.14 ± 0.12± 0.04 ± 0.01 ± (mol mol⁻¹) 0.00^(e) 0.00^(f) 0.00^(e, g) 0.00^(f, g)

Table 8 show increased ethanol yields on sugar of an S. cerevisiaestrain expressing phosphoribulokinase (PRK) and Rubisco. Physiologicalanalysis of S. cerevisiae IMU033 expressing PRK and Rubisco and theisogenic reference strain IMU032 in anaerobic chemostat cultures, grownat a dilution rate of 0.05 h-1 on a synthetic medium (pH 5) supplementedwith 12.5 g 1-1 glucose and 12.5 g 1-1 galactose as carbon sources. Toassess the impact of CO₂ concentration, chemostat cultures were runsparged either with pure nitrogen gas or with a blend of 10% CO2 and 90%nitrogen. Results are represented as average±mean deviations of datafrom independent duplicate chemostat experiments. Data pairs labelledwith the same subscripts (a,a, b,b, etc.) are considered statisticallydifferent in a standard t-test (p<0.02).

Expression of Rubisco and the four chaperones without co-expression ofPRK (strain IMC014) did not result in decreased glycerol yield (0.13 molmol⁻¹) compared to the reference strain IMU032 (0.12 mol mol⁻¹) incarbon-limited chemostat cultures supplemented with CO₂, indicating thatexpression of a phosphoribulokinase (PRK) gene is required for thefunctional pathway in S. cerevisiae to decrease glycerol production. Thephysiological impact of expression of PRK and Rubisco on growth,substrate consumption and product formation in galactose-grown anaerobicbatch cultures of S. cerevisiae was also investigated and compared withan isogenic reference strain. Growth conditions: T=30° C., pH 5.0, 10%CO₂ in inlet gas. Two independent replicate experiments were carriedout, whose growth kinetic parameters differed by less than 5%. Ethanolyield on galactose was 8% higher and glycerol production was reduced by60% in the yeast cell in which PRK and Rubisco were functionallyexpressed, compared to the yeast cell lacking these enzymes. Thedifferences were statistically significant (standard t-test (pvalue<0.02). The activities of phosphoribulokinase and of Rubisco incell extracts of the engineered strain IMU033 (table 7) enable the useof CO₂ as an electron acceptor. The ethanol yields and glycerol yieldsof strain IMU033 relative to the reference strain IMU032 (table 8) showthat this is possible in an anaerobic fermentation with increasedethanol production.

Sequences

SEQUENCE ID NO 1: Rubisco cbbM gene (synthetic; based on cbbM genefrom Thiobacillus denitrificans- pBTWW002, codon optimized Source: Hernandez et al 1996, GenBank ID: L37437.2)ATGGATCAATCTGCAAGATATGCTGACTTGTCTTTAAAGGAAGAAGATTTGATTAAAGGTGGTAGACATATTTTGGTTGCTTACAAAATGAAACCAAAATCTGGTTATGGTTATTTGGAAGCTGCTGCTCATTTTGCTGCTGAATCTTCTACAGGTACAAATGTTGAAGTTTCTACTACAGATGATTTTACAAAAGGTGTTGATGCTTTAGTTTACTACATCGATGAAGCTTCAGAAGATATGAGAATTGCTTATCCATTGGAATTATTCGACAGAAATGTTACTGACGGAAGATTCATGTTAGTTTCTTTTTTGACTTTGGCTATTGGTAACAATCAAGGAATGGGAGATATAGAACATGCAAAAATGATAGATTTTTACGTTCCAGAAAGATGTATTCAAATGTTTGATGGTCCAGCTACAGATATTTCTAATTTGTGGAGAATTTTGGGTAGACCAGTAGTTAATGGTGGTTATATTGCTGGTACTATTATTAAGCCAAAATTGGGTTTAAGACCAGAACCATTTGCTAAAGCTGCTTATCAATTTTGGTTGGGTGGAGATTTTATCAAGAATGACGAACCACAAGGTAATCAAGTTTTTTGTCCATTGAAAAAAGTTTTGCCATTGGTTTACGATGCTATGAAAAGAGCACAAGATGATACTGGTCAAGCAAAATTGTTTTCTATGAATATTACTGCAGACGATCATTATGAAATGTGTGCAAGAGCTGATTATGCTTTGGAAGTTTTCGGTCCAGATGCAGATAAATTGGCTTTTTTGGTAGATGGTTACGTTGGAGGTCCAGGAATGGTTACTACTGCTAGAAGGCAATATCCTGGTCAATATTTGCATTATCATAGAGCAGGTCACGGTGCTGTTACTTCTCCATCTGCTAAAAGAGGTTATACTGCTTTTGTTTTGGCTAAAATGTCTAGATTGCAAGGCGCTTCAGGTATTCATGTTGGTACTATGGGTTATGGAAAAATGGAAGGAGAAGGCGACGATAAGATTATTGCTTATATGATAGAAAGGGACGAATGTCAAGGTCCAGTTTATTTTCAAAAATGGTACGGTATGAAACCAACTACTCCAATTATCTCCGGAGGAATGAATGCTTTGAGATTGCCTGGTTTTTTCGAAAATTTGGGTCATGGTAACGTTATTAATACTGCAGGTGGTGGTTCTTACGGTCATATTGATTCTCCTGCTGCTGGTGCTATTTCTTTGAGACAATCTTACGAATGTTGGAAACAAGGTGCAGATCCAATTGAATTTGCTAAGGAACATAAGGAATTTGCAAGAGCTTTTGAATCTTTTCCAAAAGATGCTGATAAGTTATTTCCAGGATGGAGAGAAAAATTGGGAGTTCATTCTTAA SEQUENCE ID NO 2:Translated protein sequence of cbbM gene from Thiobacillus denitrificansMDQSARYADLSLKEEDLIKGGRHILVAYKMKPKSGYGYLEAAAHFAAESSTGTNVEVSTTDDFTKGVDALVYYIDEASEDMRIAYPLELFDRNVTDGRFMLVSFLTLAIGNNQGMGDIEHAKMIDFYVPERCIQMFDGPATDISNLWRILGRPVVNGGYIAGTIIKPKLGLRPEPFAKAAYQFWLGGDFIKNDEPQGNQVFCPLKKVLPLVYDAMKRAQDDTGQAKLFSMNITADDHYEMCARADYALEVFGPDADKLAFLVDGYVGGPGMVTTARRQYPGQYLHYHRAGHGAVTSPSAKRGYTAFVLAKMSRLQGASGIHVGTMGYGKMEGEGDDKIIAYMIERDECQGPVYFQKWYGMKPTTPIISGGMNALRLPGFFENLGHGNVINTAGGGSYGHIDSPAAGAISLRQSYECWKQGADPIEFAKEHKEFARAFESFPKDADKLFPGWREKLGVHS SEQUENCE ID NO 3:prk gene from Spinacea oleracea- pBTWW001,plasmid constructed using restriction andligation. Source: Milanez and Mural 1988, GenBank ID: M21338.1ATGTCACAACAACAAACAATTGTGATTGGTTTAGCAGCAGATTCAGGTTGTGGTAAGAGTACATTCATGAGGAGGTTAACAAGTGTTTTCGGTGGCGCGGCCGAGCCACCAAAGGGTGGTAACCCAGATTCAAACACATTGATTAGTGACACTACTACTGTTATCTGTTTGGATGATTTTCATTCCCTTGATAGAAATGGCAGGAAAGTGGAAAAAGTTACTGCTTTAGACCCAAAAGCTAATGATTTTGATCTTATGTATGAACAAGTTAAGGCTTTGAAAGAAGGTAAAGCTGTTGATAAACCTATTTATAATCATGTTTCTGGTTTGTTGGACCCTCCTGAGCTTATTCAACCTCCTAAGATCTTGGTCATTGAAGGGTTACACCCCATGTATGACGCACGTGTGAGGGAATTGCTAGACTTCAGCATCTACTTGGACATTAGCAATGAAGTTAAATTTGCCTGGAAAATTCAGAGAGACATGAAAGAAAGAGGACACAGTCTTGAAAGCATCAAAGCCAGTATTGAATCCAGAAAGCCAGATTTTGATGCTTACATTGACCCACAAAAGCAGCATGCTGATGTAGTGATTGAAGTATTGCCAACTGAACTCATTCCTGATGATGATGAAGGCAAAGTGTTGAGAGTAAGGATGATTCAGAAAGAAGGAGTCAAGTTTTTCAACCCAGTTTACTTGTTTGATGAAGGATCTACCATTTCATGGATTCCATGTGGTAGAAAATTAACATGTTCTTACCCTGGTATCAAATTTTCCTATGGCCCAGACACCTTCTATGGCAACGAGGTGACAGTAGTAGAGATGGATGGGATGTTTGACAGATTAGACGAACTAATCTACGTCGAAAGCCATTTGAGCAATCTATCAACCAAGTTTTATGGTGAAGTCACTCAACAAATGTTGAAGCACCAAAATTTCCCAGGAAGCAACAATGGAACTGGTTTCTTCCAAACCATAATTGGATTGAAGATCAGAGACTTGTTCGAGCAGCTCGTTGCTAGCAGGTCTACAGCAACTGCAACAGCTGCTAAAGCC TAG SEQUENCE ID NO 4:Translated protein sequence of prk gene from Spinacea oleraceaMSQQQTIVIGLAADSGCGKSTFMRRLTSVFGGAAEPPKGGNPDSNTLISDTTTVICLDDFHSLDRNGRKVEKVTALDPKANDFDLMYEQVKALKEGKAVDKPIYNHVSGLLDPPELIQPPKILVIEGLHPMYDARVRELLDFSIYLDISNEVKFAWKIQRDMKERGHSLESIKASIESRKPDFDAYIDPQKQHADVVIEVLPTELIPDDDEGKVLRVRMIQKEGVKFFNPVYLFDEGSTISWIPCGRKLTCSYPGIKFSYGPDTFYGNEVTVVEMDGMFDRLDELIYVESHLSNLSTKFYGEVTQQMLKHQNFPGSNNGTGFFQTIIGLKIRDLFE QLVASRSTATATAAKASEQUENCE ID NO 5: cbbQ2 gene (synthetic, based on cbbQ2 gene fromThiobacillus denitrificans- codon optimized,original sequence obtained from Beller et al2006, GenBank Gene ID: 3672366, Protein ID: AAZ98590.1ATGACTACTAACAAGGAACAATACAAGGTTCACCAAGAACCATACTACCAAGCTCAAGGTAGAGAAGTTCAATTGTACGAAGCTGCTTACAGAAACAGATTGCCAGTTATGGTTAAGGGTCCAACTGGTTGTGGTAAGTCTAGATTCGTTGAATACATGGCTTGGAAGTTGAACAAGCCATTGATCACTGTTGCTTGTAACGAAGACATGACTGCTTCTGACTTGGTTGGTAGATACTTGTTGGAAGCTAACGGTACTAGATGGTTGGACGGTCCATTGACTACTGCTGCTAGAATCGGTGCTATCTGTTACTTGGACGAAGTTGTTGAAGCTAGACAAGACACTACTGTTGTTATCCACCCATTGACTGACCACAGAAGAACTTTGCCATTGGACAAGAAGGGTGAATTGATCGAAGCTCACCCAGACTTCCAATTGGTTATCTCTTACAACCCAGGTTACCAATCTTTGATGAAGGACTTGAAGCAATCTACTAAGCAAAGATTCGCTGCTTTCGACTTCGACTACCCAGACGCTGCTTTGGAAACTACTATCTTGGCTAGAGAAACTGGTTTGGACGAAACTACTGCTGGTAGATTGGTTAAGATCGGTGGTGTTGCTAGAAACTTGAAGGGTCACGGTTTGGACGAAGGTATCTCTACTAGATTGTTGGTTTACGCTGCTACTTTGATGAAGGACGGTGTTGACGCTGGTGACGCTTGTAGAATGGCTTTGGTTAGACCAATCACTGACGACGCTGACATCAGAGAAACTTTGGACCACGCTATCGACGCTACTTTCGCTTAA SEQUENCE ID NO 6:Translated protein sequence of cbbQ2 gene fromThiobacillus denitrificansMTTNKEQYKVHQEPYYQAQGREVQLYEAAYRNRLPVMVKGPTGCGKSRFVEYMAWKLNKPLITVACNEDMTASDLVGRYLLEANGTRWLDGPLTTAARIGAICYLDEVVEARQDTTVVIHPLTDHRRTLPLDKKGELIEAHPDFQLVISYNPGYQSLMKDLKQSTKQRFAAFDFDYPDAALETTILARETGLDETTAGRLVKIGGVARNLKGHGLDEGISTRLLVYAATLMKDGVDAGDACRMALVRPITDDADIRETLDHAIDATFA SEQUENCE ID NO 7:cbbO2 gene (Synthetic, based on cbbO2 gene fromThiobacillus denitrificans- codon optimized,original sequence obtained from Beller et al2006, GenBank Gene ID: 3672365, Protein ID: YP_316394.1ATGGCTGCTTACTGGAAGGCTTTGGACACTAGATTCGCTCAAGTTGAAGAAGTTTTCGACGACTGTATGGCTGAAGCTTTGACTGTTTTGTCTGCTGAAGGTGTTGCTGCTTACTTGGAAGCTGGTAGAGTTATCGGTAAGTTGGGTAGAGGTGTTGAACCAATGTTGGCTTTCTTGGAAGAATGGCCATCTACTGCTCAAGCTGTTGGTGAAGCTGCTTTGCCAATGGTTATGGCTTTGATCCAAAGAATGCAAAAGTCTCCAAACGGTAAGGCTATCGCTCCATTCTTGCAAACTTTGGCTCCAGTTGCTAGAAGATTGCAATCTGCTGAACAATTGCAACACTACGTTGACGTTACTTTGGACTTCATGACTAGAACTACTGGTTCTATCCACGGTCACCACACTACTTTCCCATCTCCAGGTTTGCCAGAATTCTTCGCTCAAGCTCCAAACTTGTTGAACCAATTGACTTTGGCTGGTTTGAGAAACTGGGTTGAATACGGTATCAGAAACTACGGTACTCACCCAGAAAGACAACAAGACTACTTCTCTTTGCAATCTGCTGACGCTAGAGCTGTTTTGCAAAGAGAAAGACACGGTACTTTGTTGGTTGACGTTGAAAGAAAGTTGGACTTGTACTTGAGAGGTTTGTGGCAAGACCACGACCACTTGGTTCCATACTCTACTGCTTTCGACGAAATCAGAAAGCCAGTTCCATACTACGACAAGTTGGGTATGAGATTGCCAGACGTTTACGACGACTTGGTTTTGCCATGTCCAGCTGGTAGAGGTGGTGCTGGTGGTGAAGACGTTTTGGTTTCTGGTTTGGACAGATACAGAGCTACTTTGGCTCACATGGTTGGTCACAGAAGATGGTCTGAAGCTCAAATCGCTGACAACTGGTCTCCATTCCAAAGAATGGCTGTTGAATTCTTCGAAGACTGTAGAGTTGAAACTTTGTTGATGAGAGAATACCCAGGTTTGGCTAGAATCTTCAGAGCTTTGCACCCAAAGCCAGTTGAAGCTGCTTGTGACGGTGAAACTACTTCTTGTTTGAGACACAGATTGGCTATGTTGTCTAGAGCTTTCATCGACCCAGACCACGGTTACGCTGCTCCAGTTTTGAACGACTTCGTTGCTAGATTCCACGCTAGATTGGCTGACGGTACTTCTTCTACTTCTGAAATGGCTGACTTGGCTTTGTCTTACGTTGCTAAGACTAGAAGACCATCTGACCAATTCGCTAAGGTTCACTTCGACGACACTGTTGTTGACTACAGAGACGACAACAGACAATTGTGGAAGTTCATCGAAGAAGGTGACGAAGAAGAAGCTTTCGACGCTAAGAGAAAGATCGAACCAGGTGAAGAAATCCAAGGTTTGCCACCAAGACACTACCCAGAATGGGACTACACTTCTCAAACTTACAGACCAGACTGGGTTTCTGTTTACGAAGGTTTGCACAGATCTGGTAACGCTGGTGACATCGACAGATTGTTGGCTAAGCACGCTGCTTTGGCTAAGAGATTGAAGAAGATGTTGGACTTGTTGAAGCCACAAGACAAGGTTAGAGTTAGATACCAAGAAGAAGGTTCTGAATTGGACTTGGACGTTGCTATCAGATCTTTGATCGACTTCAAGGGTGGTGCTACTCCAGACCCAAGAATCAACATGTCTCACAGATCTGACGGTAGAGACATCGCTGTTATGTTGTTGTTGGACTTGTCTGAATCTTTGAACGAAAAGGCTGCTGGTGCTGGTCAAACTATCTTGGAATTGTCTCAAGAAGCTGTTTCTTTGTTGGCTTGGTCTATCGAAAAGTTGGGTGACCCATTCGCTATCGCTGGTTTCCACTCTAACACTAGACACGACGTTAGATACTTCCACATCAAGGGTTACTCTGAAAGATGGAACGACGACGTTAAGGCTAGATTGGCTGCTATGGAAGCTGGTTACTCTACTAGAATGGGTGCTGCTATGAGACACGCTGCTCACTACTTGTCTGCTAGACCAGCTGACAAGAAGTTGATGTTGATCTTGACTGACGGTAGACCATCTGACGTTGACGCTGCTGACGAAAGATTGTTGGTTGAAGACGCTAGACAAGCTGTTAAGGAATTGGACAGACAAGGTATCTTCGCTTACTGTATCTCTTTGGACGCTCAATTGAAGGCTGGTGCTGACGACTACGTTGCTGAAATCTTCGGTAGACAATACACTGTTATCGACAGAGTTGAAAGATTGCCAGAAAGATTGCCAGAATTGTTCATGGCTTTGACTAAGTAATranslated protein sequence of cbbO2 gene fromThiobacillus denitrificans SEQUENCE ID NO: 8MAAWKALDTRFAQVEEVFDDCMAEALTVLSAEGVAAYLEAGRVIGKLGRGVEPMLAFLEEWPSTAQAVGEAALPMVMALIQRMQKSPNGKAIAPFLQTLAPVARRLQSAEQLQHYVDVTLDFMTRTTGSIHGHHTTFPSPGLPEFFAQAPNLLNQLTLAGLRNWVEYGIRNYGTHPERQQDYFSLQSADARAVLQRERHGTLLVDVERKLDLYLRGLWQDHDHLVPYSTAFDEIRKPVPYYDKLGMRLPDVYDDLVLPCPAGRGGAGGEDVLVSGLDRYRATLAHMVGHRRWSEAQIADNWSPFQRMAVEFFEDCRVETLLMREYPGLARIFRALHPKPVEAACDGETTSCLRHRLAMLSRAFIDPDHGYAAPVLNDFVARFHARLADGTSSTSEMADLALSYVAKTRRPSDQFAKVHFDDTVVDYRDDNRQLWKFIEEGDEEEAFDAKRKIEPGEEIQGLPPRHYPEWDYTSQTYRPDWVSVYEGLHRSGNAGDIDRLLAKHAALAKRLKKMLDLLKPQDKVRVRYQEEGSELDLDVAIRSLIDFKGGATPDPRINMSHRSDGRDIAVMLLLDLSESLNEKAAGAGQTILELSQEAVSLLAWSIEKLGDPFAIAGFHSNTRHDVRYFHIKGYSERWNDDVKARLAAMEAGYSTRMGAAMRHAAHYLSARPADKKLMLILTDGRPSDVDAADERLLVEDARQAVKELDRQGIFAYCISLDAQLKAGADDYVAEIFGRQYTVIDRVERLPERLPELFMALTKGroEL gene (synthetic, based on GroEL fromE. coli- codon optimized, original sequenceobtained from Durfee et al 2008, Gene ID:6061450, Protein ID: YP_001732912.1 SEQUENCE ID NO: 9ATGGCTGCTAAGGACGTTAAGTTCGGTAACGACGCTAGAGTTAAGATGTTGAGAGGTGTTAACGTTTTGGCTGACGCTGTTAAGGTTACTTTGGGTCCAAAGGGTAGAAACGTTGTTTTGGACAAGTCTTTCGGTGCTCCAACTATCACTAAGGACGGTGTTTCTGTTGCTAGAGAAATCGAATTGGAAGACAAGTTCGAAAACATGGGTGCTCAAATGGTTAAGGAAGTTGCTTCTAAGGCTAACGACGCTGCTGGTGACGGTACTACTACTGCTACTGTTTTGGCTCAAGCTATCATCACTGAAGGTTTGAAGGCTGTTGCTGCTGGTATGAACCCAATGGACTTGAAGAGAGGTATCGACAAGGCTGTTACTGCTGCTGTTGAAGAATTGAAGGCTTTGTCTGTTCCATGTTCTGACTCTAAGGCTATCGCTCAAGTTGGTACTATCTCTGCTAACTCTGACGAAACTGTTGGTAAGTTGATCGCTGAAGCTATGGACAAGGTTGGTAAGGAAGGTGTTATCACTGTTGAAGACGGTACTGGTTTGCAAGACGAATTGGACGTTGTTGAAGGTATGCAATTCGACAGAGGTTACTTGTCTCCATACTTCATCAACAAGCCAGAAACTGGTGCTGTTGAATTGGAATCTCCATTCATCTTGTTGGCTGACAAGAAGATCTCTAACATCAGAGAAATGTTGCCAGTTTTGGAAGCTGTTGCTAAGGCTGGTAAGCCATTGTTGATCATCGCTGAAGACGTTGAAGGTGAAGCTTTGGCTACTTTGGTTGTTAACACTATGAGAGGTATCGTTAAGGTTGCTGCTGTTAAGGCTCCAGGTTTCGGTGACAGAAGAAAGGCTATGTTGCAAGACATCGCTACTTTGACTGGTGGTACTGTTATCTCTGAAGAAATCGGTATGGAATTGGAAAAGGCTACTTTGGAAGACTTGGGTCAAGCTAAGAGAGTTGTTATCAACAAGGACACTACTACTATCATCGACGGTGTTGGTGAAGAAGCTGCTATCCAAGGTAGAGTTGCTCAAATCAGACAACAAATCGAAGAAGCTACTTCTGACTACGACAGAGAAAAGTTGCAAGAAAGAGTTGCTAAGTTGGCTGGTGGTGTTGCTGTTATCAAGGTTGGTGCTGCTACTGAAGTTGAAATGAAGGAAAAGAAGGCTAGAGTTGAAGACGCTTTGCACGCTACTAGAGCTGCTGTTGAAGAAGGTGTTGTTGCTGGTGGTGGTGTTGCTTTGATCAGAGTTGCTTCTAAGTTGGCTGACTTGAGAGGTCAAAACGAAGACCAAAACGTTGGTATCAAGGTTGCTTTGAGAGCTATGGAAGCTCCATTGAGACAAATCGTTTTGAACTGTGGTGAAGAACCATCTGTTGTTGCTAACACTGTTAAGGGTGGTGACGGTAACTACGGTTACAACGCTGCTACTGAAGAATACGGTAACATGATCGACATGGGTATCTTGGACCCAACTAAGGTTACTAGATCTGCTTTGCAATACGCTGCTTCTGTTGCTGGTTTGATGATCACTACTGAATGTATGGTTACTGACTTGCCAAAGAACGACGCTGCTGACTTGGGTGCTGCTGGTGGTATGGGTGGTATGGGTGGTATG GGTGGTATGATGTAATranslated protein sequence of GroEL gene from E. coliSEQUENCE ID NO: 10 MAAKDVKFGNDARVKMLRGVNVLADAVKVTLGPKGRNVVLDKSFGAPTITKDGVSVAREIELEDKFENMGAQMVKEVASKANDAAGDGTTTATVLAQAIITEGLKAVAAGMNPMDLKRGIDKAVTAAVEELKALSVPCSDSKAIAQVGTISANSDETVGKLIAEAMDKVGKEGVITVEDGTGLQDELDVVEGMQFDRGYLSPYFINKPETGAVELESPFILLADKKISNIREMLPVLEAVAKAGKPLLIIAEDVEGEALATLVVNTMRGIVKVAAVKAPGFGDRRKAMLQDIATLTGGTVISEEIGMELEKATLEDLGQAKRVVINKDTTTIIDGVGEEAAIQGRVAQIRQQIEEATSDYDREKLQERVAKLAGGVAVIKVGAATEVEMKEKKARVEDALHATRAAVEEGVVAGGGVALIRVASKLADLRGQNEDQNVGIKVALRAMEAPLRQIVLNCGEEPSVVANTVKGGDGNYGYNAATEEYGNMIDMGILDPTKVTRSALQYAASVAGLMITTECMVTDLPKND AADLGAAGGMGGMGGMGGMMGroES gene (synthetic, based on GroES E. coli-codon optimized, original sequence obtained fromDurfee et al 2008, Gene ID: 6061370, Protein ID:  YP_001732911.1SEQUENCE ID NO: 11 ATGAACATCAGACCATTGCACGACAGAGTTATCGTTAAGAGAAAGGAAGTTGAAACTAAGTCTGCTGGTGGTATCGTTTTGACTGGTTCTGCTGCTGCTAAGTCTACTAGAGGTGAAGTTTTGGCTGTTGGTAACGGTAGAATCTTGGAAAACGGTGAAGTTAAGCCATTGGACGTTAAGGTTGGTGACATCGTTATCTTCAACGACGGTTACGGTGTTAAGTCTGAAAAGATCGACAACGAAGAAGTTTTGATCATGTCTGAATCTGACATCTTGGCTATCGTTGAA GCTTAATranslated protein sequence of GroES gene from E. coliSEQUENCE ID NO: 12 MNIRPLHDRVIVKRKEVETKSAGGIVLTGSAAAKSTRGEVLAVGNGRILENGEVKPLDVKVGDIVIFNDGYGVKSEKIDNEEVLIMSESDILAIVE A

1. A recombinant yeast cell functionally expressing one or morerecombinant heterologous, nucleic acid sequences encodingribulose-1,5-biphosphate carboxylase oxygenase (Rubisco) andphosphoribulokinase (PRK).
 2. A recombinant yeast cell according toclaim 1, wherein said yeast cell further comprises one or moreprokaryotic molecular chaperones.
 3. A recombinant yeast cell accordingto claim 1, wherein said chaperones are selected from the groupconsisting of GroEL, GroES, functional homologues of GroEL andfunctional homologues of GroES.
 4. A recombinant yeast cell according toclaim 1, wherein said Rubisco is a single subunit Rubisco.
 5. Arecombinant yeast cell according to claim 1, wherein said Rubisco is aprokaryotic form-II Rubisco.
 6. A recombinant yeast cell according toclaim 1, wherein said yeast cell is selected from the group consistingof Saccharomyceraceae, Schizosaccharomyces, Torulaspora, Kluyveromyces,Pichia, Zygosaccharomyces, Brettanomyces, Metschnikowia, Issatchenkia,Kloeckera, Aureobasidium.
 7. A recombinant yeast cell according to claim6, wherein the yeast cell is selected from the group ofSaccharomyceraceae.
 8. A recombinant yeast cell according to claim 7,wherein the yeast cell is selected from the group consisting ofSaccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomycesbeticus, Saccharomyces fermentati, Saccharomyces paradoxus,Saccharomyces uvarum and Saccharomyces bayanus
 9. A recombinant yeastcell according to claim 1, wherein the PRK is a PRK originating from aeukaryote.
 10. A recombinant yeast cell according to claim 9, whereinthe PRK originates from a Caryophyllales plant.
 11. A recombinant yeastcell according to claim 1, wherein the Rubisco has an activity, definedby the rate of ribulose-1,5-bisphosphate-dependent ¹⁴C-bicarbonateincorporation by cell extracts, of at least 1 nmol.min⁻¹ (mg protein)(at 30° C.).
 12. One or more vectors for the functional expression of aheterologous polypeptide in a yeast cell, wherein said vector or vectorscomprise one or more heterologous nucleic acid sequence encoding Rubiscoand PRK, wherein said Rubisco exhibits activity of carbon fixation. 13.A method for preparing an alcohol, organic acid or amino acid,comprising fermenting a carbon source with a yeast cell according toclaim 1, thereby forming the alcohol, organic acid or amino acid,wherein the yeast cell is present in a reaction medium.
 14. A methodaccording to claim 13, wherein the reaction medium comprises carbondioxide wherein the carbon dioxide concentration in the reaction mediumis at least 5% of the carbon dioxide saturation concentration. 15.Method according to claim 13, wherein ethanol is formed.
 16. (canceled)17. (canceled)
 18. (canceled)
 19. (canceled)
 20. A recombinant yeastcell according to claim 2, wherein said chaperone originates from abacterium.
 21. A recombinant yeast cell according to claim 20, whereinsaid bacterium is Escherichia coli (E. coli).
 22. A recombinant yeastcell according to claim 10, wherein the PRK originates fromAmaranthaceae or Spinacia.
 23. A method according to claim 13, whereinthe a carbon source is a carbohydrate
 24. A method according to claim14, wherein the carbon dioxide concentration in the reaction medium isat least 10% or 20% of the carbon dioxide saturation concentration.